<?xml version="1.0" encoding="utf-8"?>
<!--XBRL document created by Certent Disclosure Management 25.4.0.1 -->
<!--Based on XBRL 2.1-->
<!--Created on: 5/14/2026 1:09:10 PM-->
<!--Modified on: 5/14/2026 1:09:10 PM-->
<linkbase xmlns:link="http://www.xbrl.org/2003/linkbase" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:xbrldt="http://xbrl.org/2005/xbrldt" xsi:schemaLocation="http://www.xbrl.org/2003/linkbase http://www.xbrl.org/2003/xbrl-linkbase-2003-12-31.xsd" xmlns="http://www.xbrl.org/2003/linkbase">
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DocumentCoverPage" roleURI="http://www.heartflow.com/role/DocumentCoverPage" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#StatementCondensedConsolidatedStatementsOfRedeemableConvertiblePreferredStockAndStockholdersEquityDeficit" roleURI="http://www.heartflow.com/role/StatementCondensedConsolidatedStatementsOfRedeemableConvertiblePreferredStockAndStockholdersEquityDeficit" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DisclosureFairValueMeasurementTables" roleURI="http://www.heartflow.com/role/DisclosureFairValueMeasurementTables" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DisclosureBusinessOverviewNarrativeDetails" roleURI="http://www.heartflow.com/role/DisclosureBusinessOverviewNarrativeDetails" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DisclosureSummaryOfSignificantAccountingPoliciesNarrativeDetails" roleURI="http://www.heartflow.com/role/DisclosureSummaryOfSignificantAccountingPoliciesNarrativeDetails" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DisclosureRevenueAndContractBalancesScheduleOfTotalRevenueFromCustomersByGeographicRegionDetails" roleURI="http://www.heartflow.com/role/DisclosureRevenueAndContractBalancesScheduleOfTotalRevenueFromCustomersByGeographicRegionDetails" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DisclosureFairValueMeasurementScheduleOfFinancialAssetsAndLiabilitiesMeasuredAtFairValueOnRecurringBasisDetails" roleURI="http://www.heartflow.com/role/DisclosureFairValueMeasurementScheduleOfFinancialAssetsAndLiabilitiesMeasuredAtFairValueOnRecurringBasisDetails" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DisclosureFairValueMeasurementScheduleOfSignificantUnobservableInputsLevel3AndChangeInFairValueDetails" roleURI="http://www.heartflow.com/role/DisclosureFairValueMeasurementScheduleOfSignificantUnobservableInputsLevel3AndChangeInFairValueDetails" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DisclosureBalanceSheetComponentsNarrativeDetails" roleURI="http://www.heartflow.com/role/DisclosureBalanceSheetComponentsNarrativeDetails" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DisclosureBalanceSheetComponentsSummaryOfAvailableForSaleInvestmentsAndRestrictedCashDetails" roleURI="http://www.heartflow.com/role/DisclosureBalanceSheetComponentsSummaryOfAvailableForSaleInvestmentsAndRestrictedCashDetails" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DisclosureBalanceSheetComponentsScheduleOfAvailableForSaleInvestmentsInUnrealizedLossPositionDetails" roleURI="http://www.heartflow.com/role/DisclosureBalanceSheetComponentsScheduleOfAvailableForSaleInvestmentsInUnrealizedLossPositionDetails" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DisclosureBalanceSheetComponentsScheduleOfPropertyAndEquipmentNetDetails" roleURI="http://www.heartflow.com/role/DisclosureBalanceSheetComponentsScheduleOfPropertyAndEquipmentNetDetails" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DisclosureLeasesNarrativeDetails" roleURI="http://www.heartflow.com/role/DisclosureLeasesNarrativeDetails" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DisclosureCommitmentAndContingenciesNarrativeDetails" roleURI="http://www.heartflow.com/role/DisclosureCommitmentAndContingenciesNarrativeDetails" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DisclosureTermLoan2024CreditAgreementDetails" roleURI="http://www.heartflow.com/role/DisclosureTermLoan2024CreditAgreementDetails" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DisclosureTermLoanPrepaymentTermsAndOtherFeesDetails" roleURI="http://www.heartflow.com/role/DisclosureTermLoanPrepaymentTermsAndOtherFeesDetails" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DisclosureTermLoanInterestDetails" roleURI="http://www.heartflow.com/role/DisclosureTermLoanInterestDetails" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DisclosureTermLoanDebtIssuanceCostsAndDebtDiscountDetails" roleURI="http://www.heartflow.com/role/DisclosureTermLoanDebtIssuanceCostsAndDebtDiscountDetails" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DisclosureConvertibleNotesNarrativeDetails" roleURI="http://www.heartflow.com/role/DisclosureConvertibleNotesNarrativeDetails" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DisclosureRedeemableConvertiblePreferredStockNarrativeDetails" roleURI="http://www.heartflow.com/role/DisclosureRedeemableConvertiblePreferredStockNarrativeDetails" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DisclosureStockholdersEquityScheduleOfCommonStockReserveForIssuanceOnConvertedBasisDetails" roleURI="http://www.heartflow.com/role/DisclosureStockholdersEquityScheduleOfCommonStockReserveForIssuanceOnConvertedBasisDetails" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DisclosureCommonStockWarrantLiabilityNarrativeDetails" roleURI="http://www.heartflow.com/role/DisclosureCommonStockWarrantLiabilityNarrativeDetails" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DisclosureDerivativeLiabilityNarrativeDetails" roleURI="http://www.heartflow.com/role/DisclosureDerivativeLiabilityNarrativeDetails" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DisclosureDerivativeLiabilityScheduleOfAssumptionsUsedInDeterminingFairValueDerivativeLiabilityDetails" roleURI="http://www.heartflow.com/role/DisclosureDerivativeLiabilityScheduleOfAssumptionsUsedInDeterminingFairValueDerivativeLiabilityDetails" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DisclosureStockBasedCompensationPlansNarrativeDetails" roleURI="http://www.heartflow.com/role/DisclosureStockBasedCompensationPlansNarrativeDetails" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DisclosureStockBasedCompensationPlansSummaryOfRsuActivityDetails" roleURI="http://www.heartflow.com/role/DisclosureStockBasedCompensationPlansSummaryOfRsuActivityDetails" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DisclosureStockBasedCompensationPlansSummaryOfEstimatedFairValueOfStockOptionsDetails" roleURI="http://www.heartflow.com/role/DisclosureStockBasedCompensationPlansSummaryOfEstimatedFairValueOfStockOptionsDetails" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DisclosureStockBasedCompensationPlansScheduleOfStockBasedCompensationExpenseDetails" roleURI="http://www.heartflow.com/role/DisclosureStockBasedCompensationPlansScheduleOfStockBasedCompensationExpenseDetails" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:roleRef xlink:type="simple" xlink:href="htfl-20260331.xsd#DisclosureNetLossPerShareScheduleOfOutstandingSharesOfPotentiallyDilutiveSecuritiesDetails" roleURI="http://www.heartflow.com/role/DisclosureNetLossPerShareScheduleOfOutstandingSharesOfPotentiallyDilutiveSecuritiesDetails" xmlns="http://www.xbrl.org/2003/linkbase"></link:roleRef>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/all" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#all"></link:arcroleRef>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#hypercube-dimension"></link:arcroleRef>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-default"></link:arcroleRef>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-domain"></link:arcroleRef>
  <link:arcroleRef arcroleURI="http://xbrl.org/int/dim/arcrole/domain-member" xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#domain-member"></link:arcroleRef>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DocumentCoverPage" xlink:type="extended" xlink:title="00010 - Document - Cover Page" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityAddressesTable" xlink:label="dei_EntityAddressesTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityAddressesAddressTypeAxis" xlink:label="dei_EntityAddressesAddressTypeAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="dei_EntityAddressesTable" xlink:to="dei_EntityAddressesAddressTypeAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_AddressTypeDomain" xlink:label="dei_AddressTypeDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="dei_EntityAddressesAddressTypeAxis" xlink:to="dei_AddressTypeDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_AddressTypeDomain" xlink:label="dei_AddressTypeDomain_639143609432370955"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="dei_EntityAddressesAddressTypeAxis" xlink:to="dei_AddressTypeDomain_639143609432370955" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_FormerAddressMember" xlink:label="dei_FormerAddressMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="dei_AddressTypeDomain" xlink:to="dei_FormerAddressMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityAddressesLineItems" xlink:label="dei_EntityAddressesLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_EntityAddressesTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_DocumentType" xlink:label="dei_DocumentType"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_DocumentType" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_DocumentQuarterlyReport" xlink:label="dei_DocumentQuarterlyReport"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_DocumentQuarterlyReport" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_DocumentPeriodEndDate" xlink:label="dei_DocumentPeriodEndDate"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_DocumentPeriodEndDate" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_DocumentFiscalYearFocus" xlink:label="dei_DocumentFiscalYearFocus"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_DocumentFiscalYearFocus" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_DocumentFiscalPeriodFocus" xlink:label="dei_DocumentFiscalPeriodFocus"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_DocumentFiscalPeriodFocus" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_DocumentTransitionReport" xlink:label="dei_DocumentTransitionReport"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_DocumentTransitionReport" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityFileNumber" xlink:label="dei_EntityFileNumber"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_EntityFileNumber" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityRegistrantName" xlink:label="dei_EntityRegistrantName"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_EntityRegistrantName" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityIncorporationStateCountryCode" xlink:label="dei_EntityIncorporationStateCountryCode"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="10" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_EntityIncorporationStateCountryCode" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityTaxIdentificationNumber" xlink:label="dei_EntityTaxIdentificationNumber"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="11" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_EntityTaxIdentificationNumber" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityAddressAddressLine1" xlink:label="dei_EntityAddressAddressLine1"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="12" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_EntityAddressAddressLine1" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityAddressCityOrTown" xlink:label="dei_EntityAddressCityOrTown"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="13" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_EntityAddressCityOrTown" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityAddressStateOrProvince" xlink:label="dei_EntityAddressStateOrProvince"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="14" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_EntityAddressStateOrProvince" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityAddressPostalZipCode" xlink:label="dei_EntityAddressPostalZipCode"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="15" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_EntityAddressPostalZipCode" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_CityAreaCode" xlink:label="dei_CityAreaCode"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="16" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_CityAreaCode" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_LocalPhoneNumber" xlink:label="dei_LocalPhoneNumber"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="17" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_LocalPhoneNumber" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_Security12bTitle" xlink:label="dei_Security12bTitle"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="18" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_Security12bTitle" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_TradingSymbol" xlink:label="dei_TradingSymbol"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="19" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_TradingSymbol" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_SecurityExchangeName" xlink:label="dei_SecurityExchangeName"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="20" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_SecurityExchangeName" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityCurrentReportingStatus" xlink:label="dei_EntityCurrentReportingStatus"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="21" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_EntityCurrentReportingStatus" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityInteractiveDataCurrent" xlink:label="dei_EntityInteractiveDataCurrent"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="22" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_EntityInteractiveDataCurrent" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityFilerCategory" xlink:label="dei_EntityFilerCategory"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="23" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_EntityFilerCategory" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntitySmallBusiness" xlink:label="dei_EntitySmallBusiness"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="24" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_EntitySmallBusiness" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityEmergingGrowthCompany" xlink:label="dei_EntityEmergingGrowthCompany"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="25" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_EntityEmergingGrowthCompany" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityExTransitionPeriod" xlink:label="dei_EntityExTransitionPeriod"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="26" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_EntityExTransitionPeriod" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityShellCompany" xlink:label="dei_EntityShellCompany"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="27" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_EntityShellCompany" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityCommonStockSharesOutstanding" xlink:label="dei_EntityCommonStockSharesOutstanding"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="28" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_EntityCommonStockSharesOutstanding" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_CurrentFiscalYearEndDate" xlink:label="dei_CurrentFiscalYearEndDate"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="29" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_CurrentFiscalYearEndDate" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_EntityCentralIndexKey" xlink:label="dei_EntityCentralIndexKey"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="30" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_EntityCentralIndexKey" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/dei/2025/dei-2025.xsd#dei_AmendmentFlag" xlink:label="dei_AmendmentFlag"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="31" xlink:from="dei_EntityAddressesLineItems" xlink:to="dei_AmendmentFlag" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/StatementCondensedConsolidatedStatementsOfRedeemableConvertiblePreferredStockAndStockholdersEquityDeficit" xlink:type="extended" xlink:title="00300 - Statement - Condensed Consolidated Statements of Redeemable Convertible Preferred Stock and Stockholders&#8217; Equity (Deficit)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementTable" xlink:label="us-gaap_StatementTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementEquityComponentsAxis" xlink:label="us-gaap_StatementEquityComponentsAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementEquityComponentsAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EquityComponentDomain" xlink:label="us-gaap_EquityComponentDomain_639143609432446289"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain_639143609432446289" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockMember" xlink:label="us-gaap_CommonStockMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_CommonStockMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AdditionalPaidInCapitalMember" xlink:label="us-gaap_AdditionalPaidInCapitalMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_AdditionalPaidInCapitalMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AccumulatedOtherComprehensiveIncomeMember" xlink:label="us-gaap_AccumulatedOtherComprehensiveIncomeMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_AccumulatedOtherComprehensiveIncomeMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RetainedEarningsMember" xlink:label="us-gaap_RetainedEarningsMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_RetainedEarningsMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementClassOfStockAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain_639143609432446289"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain_639143609432446289" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RedeemableConvertiblePreferredStockMember" xlink:label="us-gaap_RedeemableConvertiblePreferredStockMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="us-gaap_RedeemableConvertiblePreferredStockMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementLineItems" xlink:label="us-gaap_StatementLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncreaseDecreaseInTemporaryEquityRollForward" xlink:label="us-gaap_IncreaseDecreaseInTemporaryEquityRollForward"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncreaseDecreaseInTemporaryEquityRollForward" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TemporaryEquitySharesOutstanding" xlink:label="us-gaap_TemporaryEquitySharesOutstanding"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_IncreaseDecreaseInTemporaryEquityRollForward" xlink:to="us-gaap_TemporaryEquitySharesOutstanding" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TemporaryEquityCarryingAmountAttributableToParent" xlink:label="us-gaap_TemporaryEquityCarryingAmountAttributableToParent"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_IncreaseDecreaseInTemporaryEquityRollForward" xlink:to="us-gaap_TemporaryEquityCarryingAmountAttributableToParent" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:label="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockSharesOutstanding" xlink:label="us-gaap_CommonStockSharesOutstanding"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_CommonStockSharesOutstanding" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockholdersEquity" xlink:label="us-gaap_StockholdersEquity"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_StockholdersEquity" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" xlink:label="us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised" xlink:label="us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardNetOfForfeitures" xlink:label="us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardNetOfForfeitures"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardNetOfForfeitures" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodValueRestrictedStockAwardNetOfForfeitures" xlink:label="us-gaap_StockIssuedDuringPeriodValueRestrictedStockAwardNetOfForfeitures"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_StockIssuedDuringPeriodValueRestrictedStockAwardNetOfForfeitures" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans" xlink:label="us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_StockIssuedDuringPeriodSharesEmployeeStockPurchasePlans" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodValueEmployeeStockPurchasePlan" xlink:label="us-gaap_StockIssuedDuringPeriodValueEmployeeStockPurchasePlan"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_StockIssuedDuringPeriodValueEmployeeStockPurchasePlan" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue" xlink:label="us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationRequisiteServicePeriodRecognitionValue" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OtherComprehensiveIncomeLossAvailableForSaleSecuritiesAdjustmentBeforeReclassificationAdjustmentsNetOfTax" xlink:label="us-gaap_OtherComprehensiveIncomeLossAvailableForSaleSecuritiesAdjustmentBeforeReclassificationAdjustmentsNetOfTax"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="10" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_OtherComprehensiveIncomeLossAvailableForSaleSecuritiesAdjustmentBeforeReclassificationAdjustmentsNetOfTax" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationAdjustmentNetOfTaxPortionAttributableToParent" xlink:label="us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationAdjustmentNetOfTaxPortionAttributableToParent"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="11" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationAdjustmentNetOfTaxPortionAttributableToParent" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NetIncomeLoss" xlink:label="us-gaap_NetIncomeLoss"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="12" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_NetIncomeLoss" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DisclosureFairValueMeasurementTables" xlink:type="extended" xlink:title="30403 - Disclosure - Fair Value Measurement (Tables)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationTable" xlink:label="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueByLiabilityClassAxis" xlink:label="us-gaap_FairValueByLiabilityClassAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationTable" xlink:to="us-gaap_FairValueByLiabilityClassAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationByLiabilityClassDomain" xlink:label="us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationByLiabilityClassDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_FairValueByLiabilityClassAxis" xlink:to="us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationByLiabilityClassDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationByLiabilityClassDomain" xlink:label="us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationByLiabilityClassDomain_639143609432483084"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_FairValueByLiabilityClassAxis" xlink:to="us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationByLiabilityClassDomain_639143609432483084" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_CommonStockWarrantLiabilityMember" xlink:label="htfl_CommonStockWarrantLiabilityMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationByLiabilityClassDomain" xlink:to="htfl_CommonStockWarrantLiabilityMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_ConvertibleNotesDerivativeLiabilityMember" xlink:label="htfl_ConvertibleNotesDerivativeLiabilityMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationByLiabilityClassDomain" xlink:to="htfl_ConvertibleNotesDerivativeLiabilityMember" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems" xlink:label="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems" xlink:to="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfFairValueAssetsAndLiabilitiesMeasuredOnRecurringBasisTableTextBlock" xlink:label="us-gaap_ScheduleOfFairValueAssetsAndLiabilitiesMeasuredOnRecurringBasisTableTextBlock"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems" xlink:to="us-gaap_ScheduleOfFairValueAssetsAndLiabilitiesMeasuredOnRecurringBasisTableTextBlock" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationTextBlock" xlink:label="us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationTextBlock"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems" xlink:to="us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationTextBlock" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DisclosureBusinessOverviewNarrativeDetails" xlink:type="extended" xlink:title="40101 - Disclosure - Business Overview (Narrative) (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_BusinessOverviewTable" xlink:label="htfl_BusinessOverviewTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SubsidiarySaleOfStockAxis" xlink:label="us-gaap_SubsidiarySaleOfStockAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="htfl_BusinessOverviewTable" xlink:to="us-gaap_SubsidiarySaleOfStockAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain_639143609432483084"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain_639143609432483084" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IPOMember" xlink:label="us-gaap_IPOMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="us-gaap_IPOMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_UnderwriterMember" xlink:label="htfl_UnderwriterMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="htfl_UnderwriterMember" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_BusinessOverviewLineItems" xlink:label="htfl_BusinessOverviewLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="htfl_BusinessOverviewLineItems" xlink:to="htfl_BusinessOverviewTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockholdersEquityNoteStockSplitConversionRatio1" xlink:label="us-gaap_StockholdersEquityNoteStockSplitConversionRatio1"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="htfl_BusinessOverviewLineItems" xlink:to="us-gaap_StockholdersEquityNoteStockSplitConversionRatio1" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction" xlink:label="us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="htfl_BusinessOverviewLineItems" xlink:to="us-gaap_SaleOfStockNumberOfSharesIssuedInTransaction" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SaleOfStockPricePerShare" xlink:label="us-gaap_SaleOfStockPricePerShare"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="htfl_BusinessOverviewLineItems" xlink:to="us-gaap_SaleOfStockPricePerShare" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_ProceedsFromIssuanceInitialPublicOfferingGross" xlink:label="htfl_ProceedsFromIssuanceInitialPublicOfferingGross"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="htfl_BusinessOverviewLineItems" xlink:to="htfl_ProceedsFromIssuanceInitialPublicOfferingGross" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PaymentsOfStockIssuanceCosts" xlink:label="us-gaap_PaymentsOfStockIssuanceCosts"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="htfl_BusinessOverviewLineItems" xlink:to="us-gaap_PaymentsOfStockIssuanceCosts" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RetainedEarningsAccumulatedDeficit" xlink:label="us-gaap_RetainedEarningsAccumulatedDeficit"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="htfl_BusinessOverviewLineItems" xlink:to="us-gaap_RetainedEarningsAccumulatedDeficit" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CashCashEquivalentsAndShortTermInvestments" xlink:label="us-gaap_CashCashEquivalentsAndShortTermInvestments"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="htfl_BusinessOverviewLineItems" xlink:to="us-gaap_CashCashEquivalentsAndShortTermInvestments" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DisclosureSummaryOfSignificantAccountingPoliciesNarrativeDetails" xlink:type="extended" xlink:title="40201 - Disclosure - Summary of Significant Accounting Policies (Narrative) (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_AccountingPoliciesTable" xlink:label="htfl_AccountingPoliciesTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_MajorCustomersAxis" xlink:label="srt_MajorCustomersAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="htfl_AccountingPoliciesTable" xlink:to="srt_MajorCustomersAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_NameOfMajorCustomerDomain" xlink:label="srt_NameOfMajorCustomerDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="srt_MajorCustomersAxis" xlink:to="srt_NameOfMajorCustomerDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_NameOfMajorCustomerDomain" xlink:label="srt_NameOfMajorCustomerDomain_639143609432493184"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="srt_MajorCustomersAxis" xlink:to="srt_NameOfMajorCustomerDomain_639143609432493184" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_NoCustomerMember" xlink:label="htfl_NoCustomerMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="srt_NameOfMajorCustomerDomain" xlink:to="htfl_NoCustomerMember" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CapitalizedContractCostAxis" xlink:label="us-gaap_CapitalizedContractCostAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="htfl_AccountingPoliciesTable" xlink:to="us-gaap_CapitalizedContractCostAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CapitalizedContractCostDomain" xlink:label="us-gaap_CapitalizedContractCostDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_CapitalizedContractCostAxis" xlink:to="us-gaap_CapitalizedContractCostDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CapitalizedContractCostDomain" xlink:label="us-gaap_CapitalizedContractCostDomain_639143609432493184"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_CapitalizedContractCostAxis" xlink:to="us-gaap_CapitalizedContractCostDomain_639143609432493184" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_ImplementationAndOnboardingCostsMember" xlink:label="htfl_ImplementationAndOnboardingCostsMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_CapitalizedContractCostDomain" xlink:to="htfl_ImplementationAndOnboardingCostsMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_NewSiteCommissionCostsMember" xlink:label="htfl_NewSiteCommissionCostsMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_CapitalizedContractCostDomain" xlink:to="htfl_NewSiteCommissionCostsMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="3" xlink:from="htfl_AccountingPoliciesTable" xlink:to="us-gaap_AwardTypeAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_639143609432503155"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_639143609432503155" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeStockOptionMember" xlink:label="us-gaap_EmployeeStockOptionMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_EmployeeStockOptionMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ConcentrationRiskByBenchmarkAxis" xlink:label="us-gaap_ConcentrationRiskByBenchmarkAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="4" xlink:from="htfl_AccountingPoliciesTable" xlink:to="us-gaap_ConcentrationRiskByBenchmarkAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ConcentrationRiskBenchmarkDomain" xlink:label="us-gaap_ConcentrationRiskBenchmarkDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_ConcentrationRiskByBenchmarkAxis" xlink:to="us-gaap_ConcentrationRiskBenchmarkDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ConcentrationRiskBenchmarkDomain" xlink:label="us-gaap_ConcentrationRiskBenchmarkDomain_639143609432503155"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_ConcentrationRiskByBenchmarkAxis" xlink:to="us-gaap_ConcentrationRiskBenchmarkDomain_639143609432503155" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SalesRevenueNetMember" xlink:label="us-gaap_SalesRevenueNetMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ConcentrationRiskBenchmarkDomain" xlink:to="us-gaap_SalesRevenueNetMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AccountsReceivableMember" xlink:label="us-gaap_AccountsReceivableMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ConcentrationRiskBenchmarkDomain" xlink:to="us-gaap_AccountsReceivableMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ConcentrationRiskByTypeAxis" xlink:label="us-gaap_ConcentrationRiskByTypeAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="5" xlink:from="htfl_AccountingPoliciesTable" xlink:to="us-gaap_ConcentrationRiskByTypeAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ConcentrationRiskTypeDomain" xlink:label="us-gaap_ConcentrationRiskTypeDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_ConcentrationRiskByTypeAxis" xlink:to="us-gaap_ConcentrationRiskTypeDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ConcentrationRiskTypeDomain" xlink:label="us-gaap_ConcentrationRiskTypeDomain_639143609432513169"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_ConcentrationRiskByTypeAxis" xlink:to="us-gaap_ConcentrationRiskTypeDomain_639143609432513169" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CustomerConcentrationRiskMember" xlink:label="us-gaap_CustomerConcentrationRiskMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ConcentrationRiskTypeDomain" xlink:to="us-gaap_CustomerConcentrationRiskMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_AccountingPoliciesLineItems" xlink:label="htfl_AccountingPoliciesLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="htfl_AccountingPoliciesLineItems" xlink:to="htfl_AccountingPoliciesTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NumberOfReportableSegments" xlink:label="us-gaap_NumberOfReportableSegments"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="htfl_AccountingPoliciesLineItems" xlink:to="us-gaap_NumberOfReportableSegments" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NumberOfOperatingSegments" xlink:label="us-gaap_NumberOfOperatingSegments"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="htfl_AccountingPoliciesLineItems" xlink:to="us-gaap_NumberOfOperatingSegments" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_NumberOfMajorCustomers" xlink:label="htfl_NumberOfMajorCustomers"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="htfl_AccountingPoliciesLineItems" xlink:to="htfl_NumberOfMajorCustomers" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtSecuritiesAvailableForSaleAllowanceForCreditLossNotPreviouslyRecorded" xlink:label="us-gaap_DebtSecuritiesAvailableForSaleAllowanceForCreditLossNotPreviouslyRecorded"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="htfl_AccountingPoliciesLineItems" xlink:to="us-gaap_DebtSecuritiesAvailableForSaleAllowanceForCreditLossNotPreviouslyRecorded" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtSecuritiesAvailableForSaleUnrealizedGainLoss" xlink:label="us-gaap_DebtSecuritiesAvailableForSaleUnrealizedGainLoss"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="htfl_AccountingPoliciesLineItems" xlink:to="us-gaap_DebtSecuritiesAvailableForSaleUnrealizedGainLoss" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_Investments" xlink:label="us-gaap_Investments"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="htfl_AccountingPoliciesLineItems" xlink:to="us-gaap_Investments" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ImpairmentOfLongLivedAssetsHeldForUse" xlink:label="us-gaap_ImpairmentOfLongLivedAssetsHeldForUse"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="htfl_AccountingPoliciesLineItems" xlink:to="us-gaap_ImpairmentOfLongLivedAssetsHeldForUse" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtConversionConvertedInstrumentAmount1" xlink:label="us-gaap_DebtConversionConvertedInstrumentAmount1"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="htfl_AccountingPoliciesLineItems" xlink:to="us-gaap_DebtConversionConvertedInstrumentAmount1" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities" xlink:label="us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="10" xlink:from="htfl_AccountingPoliciesLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_RevenueRecognitionCustomerContractPeriod" xlink:label="htfl_RevenueRecognitionCustomerContractPeriod"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="11" xlink:from="htfl_AccountingPoliciesLineItems" xlink:to="htfl_RevenueRecognitionCustomerContractPeriod" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CapitalizedContractCostAmortizationPeriod" xlink:label="us-gaap_CapitalizedContractCostAmortizationPeriod"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="12" xlink:from="htfl_AccountingPoliciesLineItems" xlink:to="us-gaap_CapitalizedContractCostAmortizationPeriod" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="13" xlink:from="htfl_AccountingPoliciesLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ForeignCurrencyTransactionGainLossAfterTax" xlink:label="us-gaap_ForeignCurrencyTransactionGainLossAfterTax"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="14" xlink:from="htfl_AccountingPoliciesLineItems" xlink:to="us-gaap_ForeignCurrencyTransactionGainLossAfterTax" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationAdjustmentNetOfTaxPortionAttributableToParent" xlink:label="us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationAdjustmentNetOfTaxPortionAttributableToParent"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="15" xlink:from="htfl_AccountingPoliciesLineItems" xlink:to="us-gaap_OtherComprehensiveIncomeForeignCurrencyTransactionAndTranslationAdjustmentNetOfTaxPortionAttributableToParent" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DisclosureRevenueAndContractBalancesScheduleOfTotalRevenueFromCustomersByGeographicRegionDetails" xlink:type="extended" xlink:title="40302 - Disclosure - Revenue and Contract Balances (Schedule of Total Revenue From Customers By Geographic Region) (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DisaggregationOfRevenueTable" xlink:label="us-gaap_DisaggregationOfRevenueTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_StatementGeographicalAxis" xlink:label="srt_StatementGeographicalAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_DisaggregationOfRevenueTable" xlink:to="srt_StatementGeographicalAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_SegmentGeographicalDomain" xlink:label="srt_SegmentGeographicalDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="srt_StatementGeographicalAxis" xlink:to="srt_SegmentGeographicalDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_SegmentGeographicalDomain" xlink:label="srt_SegmentGeographicalDomain_639143609432543361"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="srt_StatementGeographicalAxis" xlink:to="srt_SegmentGeographicalDomain_639143609432543361" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.sec.gov/country/2025/country-2025.xsd#country_US" xlink:label="country_US"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="srt_SegmentGeographicalDomain" xlink:to="country_US" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NonUsMember" xlink:label="us-gaap_NonUsMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="srt_SegmentGeographicalDomain" xlink:to="us-gaap_NonUsMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DisaggregationOfRevenueLineItems" xlink:label="us-gaap_DisaggregationOfRevenueLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_DisaggregationOfRevenueLineItems" xlink:to="us-gaap_DisaggregationOfRevenueTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" xlink:label="us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_DisaggregationOfRevenueLineItems" xlink:to="us-gaap_RevenueFromContractWithCustomerExcludingAssessedTax" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DisclosureFairValueMeasurementScheduleOfFinancialAssetsAndLiabilitiesMeasuredAtFairValueOnRecurringBasisDetails" xlink:type="extended" xlink:title="40401 - Disclosure - Fair Value Measurement (Schedule of Financial Assets and Liabilities Measured at Fair Value on a Recurring Basis) (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationTable" xlink:label="us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FinancialInstrumentAxis" xlink:label="us-gaap_FinancialInstrumentAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationTable" xlink:to="us-gaap_FinancialInstrumentAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:label="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_FinancialInstrumentAxis" xlink:to="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:label="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_639143609432553453"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_FinancialInstrumentAxis" xlink:to="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain_639143609432553453" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_MoneyMarketFundsIncludedInCashEquivalentsMember" xlink:label="htfl_MoneyMarketFundsIncludedInCashEquivalentsMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="htfl_MoneyMarketFundsIncludedInCashEquivalentsMember" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_MoneyMarketFundsIncludedInRestrictedCashNoncurrentMember" xlink:label="htfl_MoneyMarketFundsIncludedInRestrictedCashNoncurrentMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="htfl_MoneyMarketFundsIncludedInRestrictedCashNoncurrentMember" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_USGovernmentDebtSecuritiesMember" xlink:label="us-gaap_USGovernmentDebtSecuritiesMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="us-gaap_USGovernmentDebtSecuritiesMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_USTreasurySecuritiesMember" xlink:label="us-gaap_USTreasurySecuritiesMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="us-gaap_USTreasurySecuritiesMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CorporateDebtSecuritiesMember" xlink:label="us-gaap_CorporateDebtSecuritiesMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="us-gaap_CorporateDebtSecuritiesMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AgencySecuritiesMember" xlink:label="us-gaap_AgencySecuritiesMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="us-gaap_AgencySecuritiesMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AssetBackedSecuritiesMember" xlink:label="us-gaap_AssetBackedSecuritiesMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="us-gaap_AssetBackedSecuritiesMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommercialPaperMember" xlink:label="us-gaap_CommercialPaperMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="us-gaap_CommercialPaperMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:label="us-gaap_FairValueByFairValueHierarchyLevelAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationTable" xlink:to="us-gaap_FairValueByFairValueHierarchyLevelAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain_639143609432553453"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain_639143609432553453" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueInputsLevel1Member" xlink:label="us-gaap_FairValueInputsLevel1Member"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:to="us-gaap_FairValueInputsLevel1Member" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueInputsLevel2Member" xlink:label="us-gaap_FairValueInputsLevel2Member"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:to="us-gaap_FairValueInputsLevel2Member" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems" xlink:label="us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems" xlink:to="us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AssetsFairValueDisclosure" xlink:label="us-gaap_AssetsFairValueDisclosure"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_FairValueNetDerivativeAssetLiabilityMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems" xlink:to="us-gaap_AssetsFairValueDisclosure" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DisclosureFairValueMeasurementScheduleOfSignificantUnobservableInputsLevel3AndChangeInFairValueDetails" xlink:type="extended" xlink:title="40402 - Disclosure - Fair Value Measurement (Schedule of Significant Unobservable Inputs (Level 3) and the Change in Fair Value) (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueDisclosureOfAssetAndLiabilityNotMeasuredAtFairValueTable" xlink:label="us-gaap_FairValueDisclosureOfAssetAndLiabilityNotMeasuredAtFairValueTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:label="us-gaap_FairValueByFairValueHierarchyLevelAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_FairValueDisclosureOfAssetAndLiabilityNotMeasuredAtFairValueTable" xlink:to="us-gaap_FairValueByFairValueHierarchyLevelAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueInputsLevel3Member" xlink:label="us-gaap_FairValueInputsLevel3Member"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:to="us-gaap_FairValueInputsLevel3Member" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueByLiabilityClassAxis" xlink:label="us-gaap_FairValueByLiabilityClassAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_FairValueDisclosureOfAssetAndLiabilityNotMeasuredAtFairValueTable" xlink:to="us-gaap_FairValueByLiabilityClassAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationByLiabilityClassDomain" xlink:label="us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationByLiabilityClassDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_FairValueByLiabilityClassAxis" xlink:to="us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationByLiabilityClassDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_CommonStockWarrantLiabilityMember" xlink:label="htfl_CommonStockWarrantLiabilityMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationByLiabilityClassDomain" xlink:to="htfl_CommonStockWarrantLiabilityMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_ConvertibleNotesDerivativeLiabilityMember" xlink:label="htfl_ConvertibleNotesDerivativeLiabilityMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationByLiabilityClassDomain" xlink:to="htfl_ConvertibleNotesDerivativeLiabilityMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueDisclosureAssetAndLiabilityNotMeasuredAtFairValueLineItems" xlink:label="us-gaap_FairValueDisclosureAssetAndLiabilityNotMeasuredAtFairValueLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_FairValueDisclosureAssetAndLiabilityNotMeasuredAtFairValueLineItems" xlink:to="us-gaap_FairValueDisclosureOfAssetAndLiabilityNotMeasuredAtFairValueTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationCalculationRollForward" xlink:label="us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationCalculationRollForward"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_FairValueDisclosureAssetAndLiabilityNotMeasuredAtFairValueLineItems" xlink:to="us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationCalculationRollForward" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationsRecurringBasisLiabilityValue" xlink:label="us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationsRecurringBasisLiabilityValue"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationCalculationRollForward" xlink:to="us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationsRecurringBasisLiabilityValue" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLiabilityIssues" xlink:label="us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLiabilityIssues"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationCalculationRollForward" xlink:to="us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLiabilityIssues" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLiabilityGainLossIncludedInEarnings" xlink:label="us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLiabilityGainLossIncludedInEarnings"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationCalculationRollForward" xlink:to="us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLiabilityGainLossIncludedInEarnings" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLiabilitySettlements" xlink:label="us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLiabilitySettlements"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_FairValueLiabilitiesMeasuredOnRecurringBasisUnobservableInputReconciliationCalculationRollForward" xlink:to="us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisLiabilitySettlements" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DisclosureBalanceSheetComponentsNarrativeDetails" xlink:type="extended" xlink:title="40501 - Disclosure - Balance Sheet Components (Narrative) (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" xlink:label="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:label="us-gaap_PropertyPlantAndEquipmentByTypeAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" xlink:to="us-gaap_PropertyPlantAndEquipmentByTypeAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain_639143609432578583"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain_639143609432578583" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SoftwareAndSoftwareDevelopmentCostsMember" xlink:label="us-gaap_SoftwareAndSoftwareDevelopmentCostsMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_SoftwareAndSoftwareDevelopmentCostsMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_PropertyAndEquipmentExcludingSoftwareAndSoftwareDevelopmentCostsMember" xlink:label="htfl_PropertyAndEquipmentExcludingSoftwareAndSoftwareDevelopmentCostsMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="htfl_PropertyAndEquipmentExcludingSoftwareAndSoftwareDevelopmentCostsMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" xlink:to="srt_RangeAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeMember" xlink:label="srt_RangeMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeMember" xlink:label="srt_RangeMember_639143609432578583"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember_639143609432578583" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_WeightedAverageMember" xlink:label="srt_WeightedAverageMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="srt_RangeMember" xlink:to="srt_WeightedAverageMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentLineItems" xlink:label="us-gaap_PropertyPlantAndEquipmentLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_DebtSecuritiesAvailableForSaleMatuirtyPeriod" xlink:label="htfl_DebtSecuritiesAvailableForSaleMatuirtyPeriod"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="htfl_DebtSecuritiesAvailableForSaleMatuirtyPeriod" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPosition12MonthsOrLonger" xlink:label="us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPosition12MonthsOrLonger"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPosition12MonthsOrLonger" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtSecuritiesAvailableForSaleAllowanceForCreditLoss" xlink:label="us-gaap_DebtSecuritiesAvailableForSaleAllowanceForCreditLoss"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_DebtSecuritiesAvailableForSaleAllowanceForCreditLoss" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CapitalizedComputerSoftwareAdditions" xlink:label="us-gaap_CapitalizedComputerSoftwareAdditions"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_CapitalizedComputerSoftwareAdditions" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AllocatedShareBasedCompensationExpense" xlink:label="us-gaap_AllocatedShareBasedCompensationExpense"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_AllocatedShareBasedCompensationExpense" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CapitalizedComputerSoftwareAmortization1" xlink:label="us-gaap_CapitalizedComputerSoftwareAmortization1"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_CapitalizedComputerSoftwareAmortization1" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DepreciationDepletionAndAmortization" xlink:label="us-gaap_DepreciationDepletionAndAmortization"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_DepreciationDepletionAndAmortization" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DisclosureBalanceSheetComponentsSummaryOfAvailableForSaleInvestmentsAndRestrictedCashDetails" xlink:type="extended" xlink:title="40502 - Disclosure - Balance Sheet Components (Summary of Available-for-sale Investments and Restricted Cash) (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtSecuritiesAvailableForSaleTable" xlink:label="us-gaap_DebtSecuritiesAvailableForSaleTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FinancialInstrumentAxis" xlink:label="us-gaap_FinancialInstrumentAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_DebtSecuritiesAvailableForSaleTable" xlink:to="us-gaap_FinancialInstrumentAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:label="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_FinancialInstrumentAxis" xlink:to="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_MoneyMarketFundsIncludedInCashEquivalentsMember" xlink:label="htfl_MoneyMarketFundsIncludedInCashEquivalentsMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="htfl_MoneyMarketFundsIncludedInCashEquivalentsMember" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_MoneyMarketFundsIncludedInRestrictedCashNoncurrentMember" xlink:label="htfl_MoneyMarketFundsIncludedInRestrictedCashNoncurrentMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="htfl_MoneyMarketFundsIncludedInRestrictedCashNoncurrentMember" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_USGovernmentDebtSecuritiesMember" xlink:label="us-gaap_USGovernmentDebtSecuritiesMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="us-gaap_USGovernmentDebtSecuritiesMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_USTreasurySecuritiesMember" xlink:label="us-gaap_USTreasurySecuritiesMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="us-gaap_USTreasurySecuritiesMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CorporateDebtSecuritiesMember" xlink:label="us-gaap_CorporateDebtSecuritiesMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="us-gaap_CorporateDebtSecuritiesMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AgencySecuritiesMember" xlink:label="us-gaap_AgencySecuritiesMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="us-gaap_AgencySecuritiesMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AssetBackedSecuritiesMember" xlink:label="us-gaap_AssetBackedSecuritiesMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="us-gaap_AssetBackedSecuritiesMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommercialPaperMember" xlink:label="us-gaap_CommercialPaperMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="us-gaap_CommercialPaperMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfAvailableForSaleSecuritiesLineItems" xlink:label="us-gaap_ScheduleOfAvailableForSaleSecuritiesLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_ScheduleOfAvailableForSaleSecuritiesLineItems" xlink:to="us-gaap_DebtSecuritiesAvailableForSaleTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AvailableForSaleDebtSecuritiesAmortizedCostBasis" xlink:label="us-gaap_AvailableForSaleDebtSecuritiesAmortizedCostBasis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ScheduleOfAvailableForSaleSecuritiesLineItems" xlink:to="us-gaap_AvailableForSaleDebtSecuritiesAmortizedCostBasis" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedGainBeforeTax" xlink:label="us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedGainBeforeTax"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ScheduleOfAvailableForSaleSecuritiesLineItems" xlink:to="us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedGainBeforeTax" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedLossBeforeTax" xlink:label="us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedLossBeforeTax"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ScheduleOfAvailableForSaleSecuritiesLineItems" xlink:to="us-gaap_AvailableForSaleDebtSecuritiesAccumulatedGrossUnrealizedLossBeforeTax" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CashEquivalentsAtCarryingValue" xlink:label="us-gaap_CashEquivalentsAtCarryingValue"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_ScheduleOfAvailableForSaleSecuritiesLineItems" xlink:to="us-gaap_CashEquivalentsAtCarryingValue" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShortTermInvestments" xlink:label="us-gaap_ShortTermInvestments"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_ScheduleOfAvailableForSaleSecuritiesLineItems" xlink:to="us-gaap_ShortTermInvestments" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongTermInvestments" xlink:label="us-gaap_LongTermInvestments"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_ScheduleOfAvailableForSaleSecuritiesLineItems" xlink:to="us-gaap_LongTermInvestments" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestrictedCash" xlink:label="us-gaap_RestrictedCash"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_ScheduleOfAvailableForSaleSecuritiesLineItems" xlink:to="us-gaap_RestrictedCash" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AvailableForSaleSecuritiesDebtSecurities" xlink:label="us-gaap_AvailableForSaleSecuritiesDebtSecurities"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="us-gaap_ScheduleOfAvailableForSaleSecuritiesLineItems" xlink:to="us-gaap_AvailableForSaleSecuritiesDebtSecurities" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DisclosureBalanceSheetComponentsScheduleOfAvailableForSaleInvestmentsInUnrealizedLossPositionDetails" xlink:type="extended" xlink:title="40504 - Disclosure - Balance Sheet Components (Schedule of Available-for-sale Investments in an Unrealized Loss Position) (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtSecuritiesAvailableForSaleTable" xlink:label="us-gaap_DebtSecuritiesAvailableForSaleTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FinancialInstrumentAxis" xlink:label="us-gaap_FinancialInstrumentAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_DebtSecuritiesAvailableForSaleTable" xlink:to="us-gaap_FinancialInstrumentAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:label="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_FinancialInstrumentAxis" xlink:to="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_USGovernmentDebtSecuritiesMember" xlink:label="us-gaap_USGovernmentDebtSecuritiesMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="us-gaap_USGovernmentDebtSecuritiesMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CorporateDebtSecuritiesMember" xlink:label="us-gaap_CorporateDebtSecuritiesMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="us-gaap_CorporateDebtSecuritiesMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AgencySecuritiesMember" xlink:label="us-gaap_AgencySecuritiesMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="us-gaap_AgencySecuritiesMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AssetBackedSecuritiesMember" xlink:label="us-gaap_AssetBackedSecuritiesMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="us-gaap_AssetBackedSecuritiesMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommercialPaperMember" xlink:label="us-gaap_CommercialPaperMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_TransfersAndServicingOfFinancialInstrumentsTypesOfFinancialInstrumentsDomain" xlink:to="us-gaap_CommercialPaperMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfAvailableForSaleSecuritiesLineItems" xlink:label="us-gaap_ScheduleOfAvailableForSaleSecuritiesLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_ScheduleOfAvailableForSaleSecuritiesLineItems" xlink:to="us-gaap_DebtSecuritiesAvailableForSaleTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPositionLessThan12Months" xlink:label="us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPositionLessThan12Months"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ScheduleOfAvailableForSaleSecuritiesLineItems" xlink:to="us-gaap_DebtSecuritiesAvailableForSaleContinuousUnrealizedLossPositionLessThan12Months" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPosition" xlink:label="us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPosition"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ScheduleOfAvailableForSaleSecuritiesLineItems" xlink:to="us-gaap_DebtSecuritiesAvailableForSaleUnrealizedLossPosition" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DisclosureBalanceSheetComponentsScheduleOfPropertyAndEquipmentNetDetails" xlink:type="extended" xlink:title="40507 - Disclosure - Balance Sheet Components (Schedule of Property and Equipment, Net) (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" xlink:label="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:label="us-gaap_PropertyPlantAndEquipmentByTypeAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" xlink:to="us-gaap_PropertyPlantAndEquipmentByTypeAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_ComputerEquipmentAndSoftwareMember" xlink:label="htfl_ComputerEquipmentAndSoftwareMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="htfl_ComputerEquipmentAndSoftwareMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FurnitureAndFixturesMember" xlink:label="us-gaap_FurnitureAndFixturesMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_FurnitureAndFixturesMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SoftwareAndSoftwareDevelopmentCostsMember" xlink:label="us-gaap_SoftwareAndSoftwareDevelopmentCostsMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_SoftwareAndSoftwareDevelopmentCostsMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LeaseholdImprovementsMember" xlink:label="us-gaap_LeaseholdImprovementsMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_LeaseholdImprovementsMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ConstructionInProgressMember" xlink:label="us-gaap_ConstructionInProgressMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_ConstructionInProgressMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentLineItems" xlink:label="us-gaap_PropertyPlantAndEquipmentLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentGross" xlink:label="us-gaap_PropertyPlantAndEquipmentGross"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_PropertyPlantAndEquipmentGross" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment" xlink:label="us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PropertyPlantAndEquipmentNet" xlink:label="us-gaap_PropertyPlantAndEquipmentNet"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_PropertyPlantAndEquipmentNet" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DisclosureLeasesNarrativeDetails" xlink:type="extended" xlink:title="40601 - Disclosure - Leases (Narrative) (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeLeaseDescriptionTable" xlink:label="us-gaap_LesseeLeaseDescriptionTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_LesseeLeaseDescriptionTable" xlink:to="srt_RangeAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeMember" xlink:label="srt_RangeMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="srt_RangeMember" xlink:to="srt_MinimumMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="srt_RangeMember" xlink:to="srt_MaximumMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_StatementGeographicalAxis" xlink:label="srt_StatementGeographicalAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_LesseeLeaseDescriptionTable" xlink:to="srt_StatementGeographicalAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_SegmentGeographicalDomain" xlink:label="srt_SegmentGeographicalDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="srt_StatementGeographicalAxis" xlink:to="srt_SegmentGeographicalDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_MountainViewCaliforniaMember" xlink:label="htfl_MountainViewCaliforniaMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="srt_SegmentGeographicalDomain" xlink:to="htfl_MountainViewCaliforniaMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_SantaRosaCaliforniaMember" xlink:label="htfl_SantaRosaCaliforniaMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="srt_SegmentGeographicalDomain" xlink:to="htfl_SantaRosaCaliforniaMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_SanFranciscoCaliforniaMember" xlink:label="htfl_SanFranciscoCaliforniaMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="srt_SegmentGeographicalDomain" xlink:to="htfl_SanFranciscoCaliforniaMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_RohnertParkCaliforniaMember" xlink:label="htfl_RohnertParkCaliforniaMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="srt_SegmentGeographicalDomain" xlink:to="htfl_RohnertParkCaliforniaMember" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_AustinTexasMember" xlink:label="htfl_AustinTexasMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="srt_SegmentGeographicalDomain" xlink:to="htfl_AustinTexasMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_TokyoJapanMember" xlink:label="htfl_TokyoJapanMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="srt_SegmentGeographicalDomain" xlink:to="htfl_TokyoJapanMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeLeaseDescriptionLineItems" xlink:label="us-gaap_LesseeLeaseDescriptionLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_LesseeLeaseDescriptionTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NetRentableArea" xlink:label="us-gaap_NetRentableArea"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_NetRentableArea" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_TenantImprovementAllowanceUnused" xlink:label="htfl_TenantImprovementAllowanceUnused"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="htfl_TenantImprovementAllowanceUnused" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_LesseeOperatingSubleaseLetterOfCreditProvidedInPlaceOfSecurityDeposit" xlink:label="htfl_LesseeOperatingSubleaseLetterOfCreditProvidedInPlaceOfSecurityDeposit"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="htfl_LesseeOperatingSubleaseLetterOfCreditProvidedInPlaceOfSecurityDeposit" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeOperatingLeaseTermOfContract" xlink:label="us-gaap_LesseeOperatingLeaseTermOfContract"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_LesseeOperatingLeaseTermOfContract" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AssetImpairmentCharges" xlink:label="us-gaap_AssetImpairmentCharges"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_AssetImpairmentCharges" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingLeaseImpairmentLoss" xlink:label="us-gaap_OperatingLeaseImpairmentLoss"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_OperatingLeaseImpairmentLoss" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ImpairmentOfLeasehold" xlink:label="us-gaap_ImpairmentOfLeasehold"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_ImpairmentOfLeasehold" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SecurityDeposit" xlink:label="us-gaap_SecurityDeposit"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_SecurityDeposit" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingLeaseLiability" xlink:label="us-gaap_OperatingLeaseLiability"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="10" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_OperatingLeaseLiability" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingLeaseRightOfUseAsset" xlink:label="us-gaap_OperatingLeaseRightOfUseAsset"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="11" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_OperatingLeaseRightOfUseAsset" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LesseeOperatingLeaseRenewalTerm" xlink:label="us-gaap_LesseeOperatingLeaseRenewalTerm"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="12" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_LesseeOperatingLeaseRenewalTerm" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_LesseeOperatingLeaseNumberOfRenewalOptions" xlink:label="htfl_LesseeOperatingLeaseNumberOfRenewalOptions"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="13" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="htfl_LesseeOperatingLeaseNumberOfRenewalOptions" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_LesseeOperatingLeaseMonthlyPayment" xlink:label="htfl_LesseeOperatingLeaseMonthlyPayment"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="14" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="htfl_LesseeOperatingLeaseMonthlyPayment" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_LesseeOperatingLeaseMonthlyPaymentAmendedLeaseTerm" xlink:label="htfl_LesseeOperatingLeaseMonthlyPaymentAmendedLeaseTerm"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="15" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="htfl_LesseeOperatingLeaseMonthlyPaymentAmendedLeaseTerm" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_LesseeOperatingLeaseNumberOfContracts" xlink:label="htfl_LesseeOperatingLeaseNumberOfContracts"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="16" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="htfl_LesseeOperatingLeaseNumberOfContracts" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_OperatingLeasePayments" xlink:label="us-gaap_OperatingLeasePayments"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="17" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="us-gaap_OperatingLeasePayments" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_LesseeOepratingSubleaseInitialOccupancyPercentage" xlink:label="htfl_LesseeOepratingSubleaseInitialOccupancyPercentage"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="18" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="htfl_LesseeOepratingSubleaseInitialOccupancyPercentage" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_LesseeOperatingSubleaseAnnualBaseRent" xlink:label="htfl_LesseeOperatingSubleaseAnnualBaseRent"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="19" xlink:from="us-gaap_LesseeLeaseDescriptionLineItems" xlink:to="htfl_LesseeOperatingSubleaseAnnualBaseRent" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DisclosureCommitmentAndContingenciesNarrativeDetails" xlink:type="extended" xlink:title="40701 - Disclosure - Commitment and Contingencies (Narrative) (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LossContingenciesTable" xlink:label="us-gaap_LossContingenciesTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_LossContingenciesTable" xlink:to="srt_RangeAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeMember" xlink:label="srt_RangeMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="srt_RangeMember" xlink:to="srt_MinimumMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LossContingenciesLineItems" xlink:label="us-gaap_LossContingenciesLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_LossContingenciesLineItems" xlink:to="us-gaap_LossContingenciesTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ContractualObligation" xlink:label="us-gaap_ContractualObligation"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_LossContingenciesLineItems" xlink:to="us-gaap_ContractualObligation" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RoyaltyExpense" xlink:label="us-gaap_RoyaltyExpense"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_LossContingenciesLineItems" xlink:to="us-gaap_RoyaltyExpense" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongTermPurchaseCommitmentAmount" xlink:label="us-gaap_LongTermPurchaseCommitmentAmount"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_LossContingenciesLineItems" xlink:to="us-gaap_LongTermPurchaseCommitmentAmount" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LongtermPurchaseCommitmentPeriod" xlink:label="us-gaap_LongtermPurchaseCommitmentPeriod"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_LossContingenciesLineItems" xlink:to="us-gaap_LongtermPurchaseCommitmentPeriod" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LossContingencyAccrualAtCarryingValue" xlink:label="us-gaap_LossContingencyAccrualAtCarryingValue"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_LossContingenciesLineItems" xlink:to="us-gaap_LossContingencyAccrualAtCarryingValue" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DisclosureTermLoan2024CreditAgreementDetails" xlink:type="extended" xlink:title="40801 - Disclosure - Term Loan (2024 Credit Agreement) (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentTable" xlink:label="us-gaap_DebtInstrumentTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentAxis" xlink:label="us-gaap_DebtInstrumentAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_DebtInstrumentTable" xlink:to="us-gaap_DebtInstrumentAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain_639143609432669126"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain_639143609432669126" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_CreditAgreement2024Member" xlink:label="htfl_CreditAgreement2024Member"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_DebtInstrumentNameDomain" xlink:to="htfl_CreditAgreement2024Member" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_A2025ConvertiblePromissoryNotesMember" xlink:label="htfl_A2025ConvertiblePromissoryNotesMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_DebtInstrumentNameDomain" xlink:to="htfl_A2025ConvertiblePromissoryNotesMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentLineItems" xlink:label="us-gaap_DebtInstrumentLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_DebtInstrumentTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_NotesPayable" xlink:label="us-gaap_NotesPayable"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_NotesPayable" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_NotesConverted" xlink:label="htfl_NotesConverted"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="htfl_NotesConverted" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DisclosureTermLoanPrepaymentTermsAndOtherFeesDetails" xlink:type="extended" xlink:title="40802 - Disclosure - Term Loan (Prepayment Terms and Other Fees) (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentTable" xlink:label="us-gaap_DebtInstrumentTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentAxis" xlink:label="us-gaap_DebtInstrumentAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_DebtInstrumentTable" xlink:to="us-gaap_DebtInstrumentAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_AmendedCreditAgreement2024Member" xlink:label="htfl_AmendedCreditAgreement2024Member"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_DebtInstrumentNameDomain" xlink:to="htfl_AmendedCreditAgreement2024Member" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentLineItems" xlink:label="us-gaap_DebtInstrumentLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_DebtInstrumentTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DeferredFinanceCostsNet" xlink:label="us-gaap_DeferredFinanceCostsNet"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_DeferredFinanceCostsNet" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_DebtInstrumentExitFeePercentage" xlink:label="htfl_DebtInstrumentExitFeePercentage"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="htfl_DebtInstrumentExitFeePercentage" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_DebtInstrumentEarlyRepaymentFeePercentage" xlink:label="htfl_DebtInstrumentEarlyRepaymentFeePercentage"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="htfl_DebtInstrumentEarlyRepaymentFeePercentage" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PaymentsOfDebtExtinguishmentCosts" xlink:label="us-gaap_PaymentsOfDebtExtinguishmentCosts"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_PaymentsOfDebtExtinguishmentCosts" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RepaymentsOfLongTermDebt" xlink:label="us-gaap_RepaymentsOfLongTermDebt"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_RepaymentsOfLongTermDebt" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentRepaidPrincipal" xlink:label="us-gaap_DebtInstrumentRepaidPrincipal"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_DebtInstrumentRepaidPrincipal" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_RepaymentOfAccruedInterest" xlink:label="htfl_RepaymentOfAccruedInterest"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="htfl_RepaymentOfAccruedInterest" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DisclosureTermLoanInterestDetails" xlink:type="extended" xlink:title="40803 - Disclosure - Term Loan (Interest) (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentTable" xlink:label="us-gaap_DebtInstrumentTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentAxis" xlink:label="us-gaap_DebtInstrumentAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_DebtInstrumentTable" xlink:to="us-gaap_DebtInstrumentAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_CreditAgreement2024Member" xlink:label="htfl_CreditAgreement2024Member"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_DebtInstrumentNameDomain" xlink:to="htfl_CreditAgreement2024Member" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_VariableRateAxis" xlink:label="us-gaap_VariableRateAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_DebtInstrumentTable" xlink:to="us-gaap_VariableRateAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_VariableRateDomain" xlink:label="us-gaap_VariableRateDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_VariableRateAxis" xlink:to="us-gaap_VariableRateDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_VariableRateDomain" xlink:label="us-gaap_VariableRateDomain_639143609432679209"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_VariableRateAxis" xlink:to="us-gaap_VariableRateDomain_639143609432679209" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_BaseRateMember" xlink:label="us-gaap_BaseRateMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_VariableRateDomain" xlink:to="us-gaap_BaseRateMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SecuredOvernightFinancingRateSofrMember" xlink:label="us-gaap_SecuredOvernightFinancingRateSofrMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_VariableRateDomain" xlink:to="us-gaap_SecuredOvernightFinancingRateSofrMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PrimeRateMember" xlink:label="us-gaap_PrimeRateMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_VariableRateDomain" xlink:to="us-gaap_PrimeRateMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_FederalReserveBankOfNewYorkRateMember" xlink:label="htfl_FederalReserveBankOfNewYorkRateMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_VariableRateDomain" xlink:to="htfl_FederalReserveBankOfNewYorkRateMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_OneMonthSecuredOvernightFinancingRateSOFRMember" xlink:label="htfl_OneMonthSecuredOvernightFinancingRateSOFRMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_VariableRateDomain" xlink:to="htfl_OneMonthSecuredOvernightFinancingRateSOFRMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="3" xlink:from="us-gaap_DebtInstrumentTable" xlink:to="srt_RangeAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeMember" xlink:label="srt_RangeMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="srt_RangeMember" xlink:to="srt_MaximumMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="srt_RangeMember" xlink:to="srt_MinimumMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentLineItems" xlink:label="us-gaap_DebtInstrumentLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_DebtInstrumentTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentBasisSpreadOnVariableRate1" xlink:label="us-gaap_DebtInstrumentBasisSpreadOnVariableRate1"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_DebtInstrumentBasisSpreadOnVariableRate1" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_DebtInstrumentInterestRatePaidInKind" xlink:label="htfl_DebtInstrumentInterestRatePaidInKind"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="htfl_DebtInstrumentInterestRatePaidInKind" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DisclosureTermLoanDebtIssuanceCostsAndDebtDiscountDetails" xlink:type="extended" xlink:title="40804 - Disclosure - Term Loan (Debt Issuance Costs and Debt Discount) (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentTable" xlink:label="us-gaap_DebtInstrumentTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentAxis" xlink:label="us-gaap_DebtInstrumentAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_DebtInstrumentTable" xlink:to="us-gaap_DebtInstrumentAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_A2025ConvertiblePromissoryNotesMember" xlink:label="htfl_A2025ConvertiblePromissoryNotesMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_DebtInstrumentNameDomain" xlink:to="htfl_A2025ConvertiblePromissoryNotesMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentLineItems" xlink:label="us-gaap_DebtInstrumentLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_DebtInstrumentTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_NotesConverted" xlink:label="htfl_NotesConverted"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="htfl_NotesConverted" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_DebtInstrumentProRataDebtDiscount" xlink:label="htfl_DebtInstrumentProRataDebtDiscount"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="htfl_DebtInstrumentProRataDebtDiscount" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DisclosureConvertibleNotesNarrativeDetails" xlink:type="extended" xlink:title="40901 - Disclosure - Convertible Notes (Narrative) (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LineOfCreditFacilityTable" xlink:label="us-gaap_LineOfCreditFacilityTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentAxis" xlink:label="us-gaap_DebtInstrumentAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_LineOfCreditFacilityTable" xlink:to="us-gaap_DebtInstrumentAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_A2025ConvertiblePromissoryNotesMember" xlink:label="htfl_A2025ConvertiblePromissoryNotesMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_DebtInstrumentNameDomain" xlink:to="htfl_A2025ConvertiblePromissoryNotesMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_A2025ConvertiblePromissoryNotesSettledInCashMember" xlink:label="htfl_A2025ConvertiblePromissoryNotesSettledInCashMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="htfl_A2025ConvertiblePromissoryNotesMember" xlink:to="htfl_A2025ConvertiblePromissoryNotesSettledInCashMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_A2025ConvertiblePromissoryNotesSettledInLieuOfCashMember" xlink:label="htfl_A2025ConvertiblePromissoryNotesSettledInLieuOfCashMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="htfl_A2025ConvertiblePromissoryNotesMember" xlink:to="htfl_A2025ConvertiblePromissoryNotesSettledInLieuOfCashMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_A2025ConvertiblePromissoryNotesIssuedFromConversionOfPrincipalUnder2024TermLoanMember" xlink:label="htfl_A2025ConvertiblePromissoryNotesIssuedFromConversionOfPrincipalUnder2024TermLoanMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="htfl_A2025ConvertiblePromissoryNotesMember" xlink:to="htfl_A2025ConvertiblePromissoryNotesIssuedFromConversionOfPrincipalUnder2024TermLoanMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_LineOfCreditFacilityLineItems" xlink:label="us-gaap_LineOfCreditFacilityLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_LineOfCreditFacilityLineItems" xlink:to="us-gaap_LineOfCreditFacilityTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentFaceAmount" xlink:label="us-gaap_DebtInstrumentFaceAmount"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_LineOfCreditFacilityLineItems" xlink:to="us-gaap_DebtInstrumentFaceAmount" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities" xlink:label="us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_LineOfCreditFacilityLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_DebtInstrumentIssuedOnEmployeeBasedCompensation" xlink:label="htfl_DebtInstrumentIssuedOnEmployeeBasedCompensation"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_LineOfCreditFacilityLineItems" xlink:to="htfl_DebtInstrumentIssuedOnEmployeeBasedCompensation" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtConversionConvertedInstrumentAmount1" xlink:label="us-gaap_DebtConversionConvertedInstrumentAmount1"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_LineOfCreditFacilityLineItems" xlink:to="us-gaap_DebtConversionConvertedInstrumentAmount1" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ProceedsFromConvertibleDebt" xlink:label="us-gaap_ProceedsFromConvertibleDebt"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_LineOfCreditFacilityLineItems" xlink:to="us-gaap_ProceedsFromConvertibleDebt" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DeferredFinanceCostsNet" xlink:label="us-gaap_DeferredFinanceCostsNet"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_LineOfCreditFacilityLineItems" xlink:to="us-gaap_DeferredFinanceCostsNet" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentTerm" xlink:label="us-gaap_DebtInstrumentTerm"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_LineOfCreditFacilityLineItems" xlink:to="us-gaap_DebtInstrumentTerm" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SharePrice" xlink:label="us-gaap_SharePrice"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="us-gaap_LineOfCreditFacilityLineItems" xlink:to="us-gaap_SharePrice" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_DebtInstrumentConversionPriceDiscountPercentage" xlink:label="htfl_DebtInstrumentConversionPriceDiscountPercentage"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="10" xlink:from="us-gaap_LineOfCreditFacilityLineItems" xlink:to="htfl_DebtInstrumentConversionPriceDiscountPercentage" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DerivativeLiabilities" xlink:label="us-gaap_DerivativeLiabilities"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="11" xlink:from="us-gaap_LineOfCreditFacilityLineItems" xlink:to="us-gaap_DerivativeLiabilities" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalConvertibleDebtWithConversionFeature" xlink:label="us-gaap_AdjustmentsToAdditionalPaidInCapitalConvertibleDebtWithConversionFeature"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="12" xlink:from="us-gaap_LineOfCreditFacilityLineItems" xlink:to="us-gaap_AdjustmentsToAdditionalPaidInCapitalConvertibleDebtWithConversionFeature" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DisclosureRedeemableConvertiblePreferredStockNarrativeDetails" xlink:type="extended" xlink:title="41001 - Disclosure - Redeemable Convertible Preferred Stock (Narrative) (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TemporaryEquityByClassOfStockTable" xlink:label="us-gaap_TemporaryEquityByClassOfStockTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StatementClassOfStockAxis" xlink:label="us-gaap_StatementClassOfStockAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_TemporaryEquityByClassOfStockTable" xlink:to="us-gaap_StatementClassOfStockAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockDomain" xlink:label="us-gaap_ClassOfStockDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockMember" xlink:label="us-gaap_CommonStockMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="us-gaap_CommonStockMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_SeriesARedeemableConvertiblePreferredStockMember" xlink:label="htfl_SeriesARedeemableConvertiblePreferredStockMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="htfl_SeriesARedeemableConvertiblePreferredStockMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_SeriesB1RedeemableConvertiblePreferredStockMember" xlink:label="htfl_SeriesB1RedeemableConvertiblePreferredStockMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="htfl_SeriesB1RedeemableConvertiblePreferredStockMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_SeriesB2RedeemableConvertiblePreferredStockMember" xlink:label="htfl_SeriesB2RedeemableConvertiblePreferredStockMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="htfl_SeriesB2RedeemableConvertiblePreferredStockMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_SeriesCRedeemableConvertiblePreferredStockMember" xlink:label="htfl_SeriesCRedeemableConvertiblePreferredStockMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="htfl_SeriesCRedeemableConvertiblePreferredStockMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_SeriesDRedeemableConvertiblePreferredStockMember" xlink:label="htfl_SeriesDRedeemableConvertiblePreferredStockMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="htfl_SeriesDRedeemableConvertiblePreferredStockMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_SeriesERedeemableConvertiblePreferredStockMember" xlink:label="htfl_SeriesERedeemableConvertiblePreferredStockMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="htfl_SeriesERedeemableConvertiblePreferredStockMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_SeriesFRedeemableConvertiblePreferredStockMember" xlink:label="htfl_SeriesFRedeemableConvertiblePreferredStockMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="htfl_SeriesFRedeemableConvertiblePreferredStockMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_SeriesF1RedeemableConvertiblePreferredStockMember" xlink:label="htfl_SeriesF1RedeemableConvertiblePreferredStockMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="htfl_SeriesF1RedeemableConvertiblePreferredStockMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_TemporaryEquityLineItems" xlink:label="us-gaap_TemporaryEquityLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_TemporaryEquityLineItems" xlink:to="us-gaap_TemporaryEquityByClassOfStockTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SharePrice" xlink:label="us-gaap_SharePrice"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_TemporaryEquityLineItems" xlink:to="us-gaap_SharePrice" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PaymentsOfStockIssuanceCosts" xlink:label="us-gaap_PaymentsOfStockIssuanceCosts"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_TemporaryEquityLineItems" xlink:to="us-gaap_PaymentsOfStockIssuanceCosts" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtConversionConvertedInstrumentAmount1" xlink:label="us-gaap_DebtConversionConvertedInstrumentAmount1"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_TemporaryEquityLineItems" xlink:to="us-gaap_DebtConversionConvertedInstrumentAmount1" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentFaceAmount" xlink:label="us-gaap_DebtInstrumentFaceAmount"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_TemporaryEquityLineItems" xlink:to="us-gaap_DebtInstrumentFaceAmount" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities" xlink:label="us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_TemporaryEquityLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesConversionOfConvertibleSecurities" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_DebtInstrumentConversionPriceDiscountPercentage" xlink:label="htfl_DebtInstrumentConversionPriceDiscountPercentage"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_TemporaryEquityLineItems" xlink:to="htfl_DebtInstrumentConversionPriceDiscountPercentage" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PreferredStockConvertibleConversionRatio" xlink:label="us-gaap_PreferredStockConvertibleConversionRatio"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_TemporaryEquityLineItems" xlink:to="us-gaap_PreferredStockConvertibleConversionRatio" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PreferredStockSharesIssued" xlink:label="us-gaap_PreferredStockSharesIssued"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="us-gaap_TemporaryEquityLineItems" xlink:to="us-gaap_PreferredStockSharesIssued" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PreferredStockSharesOutstanding" xlink:label="us-gaap_PreferredStockSharesOutstanding"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="10" xlink:from="us-gaap_TemporaryEquityLineItems" xlink:to="us-gaap_PreferredStockSharesOutstanding" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DisclosureStockholdersEquityScheduleOfCommonStockReserveForIssuanceOnConvertedBasisDetails" xlink:type="extended" xlink:title="41102 - Disclosure - Stockholders&apos; Equity (Schedule Of Common Stock Reserve For Issuance On Converted Basis) (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfStockByClassTable" xlink:label="us-gaap_ScheduleOfStockByClassTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfStockByClassTable" xlink:to="us-gaap_AwardTypeAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeStockOptionMember" xlink:label="us-gaap_EmployeeStockOptionMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_EmployeeStockOptionMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestrictedStockUnitsRSUMember" xlink:label="us-gaap_RestrictedStockUnitsRSUMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_RestrictedStockUnitsRSUMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_SharesReservedForIssuanceMember" xlink:label="htfl_SharesReservedForIssuanceMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="htfl_SharesReservedForIssuanceMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfStockLineItems" xlink:label="us-gaap_ClassOfStockLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_ScheduleOfStockByClassTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockCapitalSharesReservedForFutureIssuance" xlink:label="us-gaap_CommonStockCapitalSharesReservedForFutureIssuance"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_CommonStockCapitalSharesReservedForFutureIssuance" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DisclosureCommonStockWarrantLiabilityNarrativeDetails" xlink:type="extended" xlink:title="41201 - Disclosure - Common Stock Warrant Liability (Narrative) (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfWarrantOrRightTable" xlink:label="us-gaap_ClassOfWarrantOrRightTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfWarrantOrRightAxis" xlink:label="us-gaap_ClassOfWarrantOrRightAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ClassOfWarrantOrRightTable" xlink:to="us-gaap_ClassOfWarrantOrRightAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfWarrantOrRightDomain" xlink:label="us-gaap_ClassOfWarrantOrRightDomain_639143609432724357"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_ClassOfWarrantOrRightAxis" xlink:to="us-gaap_ClassOfWarrantOrRightDomain_639143609432724357" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_CommonStockWarrantMember" xlink:label="htfl_CommonStockWarrantMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ClassOfWarrantOrRightDomain" xlink:to="htfl_CommonStockWarrantMember" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfWarrantOrRightLineItems" xlink:label="us-gaap_ClassOfWarrantOrRightLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_ClassOfWarrantOrRightLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights" xlink:label="us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ClassOfWarrantOrRightLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightNumberOfSecuritiesCalledByWarrantsOrRights" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueAdjustmentOfWarrants" xlink:label="us-gaap_FairValueAdjustmentOfWarrants"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ClassOfWarrantOrRightLineItems" xlink:to="us-gaap_FairValueAdjustmentOfWarrants" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:label="us-gaap_StockIssuedDuringPeriodSharesNewIssues"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ClassOfWarrantOrRightLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesNewIssues" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_WarrantsExercisedFairValueTransferredToStockholdersEquity" xlink:label="htfl_WarrantsExercisedFairValueTransferredToStockholdersEquity"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_ClassOfWarrantOrRightLineItems" xlink:to="htfl_WarrantsExercisedFairValueTransferredToStockholdersEquity" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DisclosureDerivativeLiabilityNarrativeDetails" xlink:type="extended" xlink:title="41301 - Disclosure - Derivative Liability (Narrative) (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable" xlink:label="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentAxis" xlink:label="us-gaap_DebtInstrumentAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable" xlink:to="us-gaap_DebtInstrumentAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DebtInstrumentNameDomain" xlink:label="us-gaap_DebtInstrumentNameDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_A2025ConvertiblePromissoryNotesMember" xlink:label="htfl_A2025ConvertiblePromissoryNotesMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_DebtInstrumentNameDomain" xlink:to="htfl_A2025ConvertiblePromissoryNotesMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems" xlink:label="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems" xlink:to="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DerivativeLiabilities" xlink:label="us-gaap_DerivativeLiabilities"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems" xlink:to="us-gaap_DerivativeLiabilities" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_DebtInstrumentConversionPriceDiscountPercentage" xlink:label="htfl_DebtInstrumentConversionPriceDiscountPercentage"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems" xlink:to="htfl_DebtInstrumentConversionPriceDiscountPercentage" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DisclosureDerivativeLiabilityScheduleOfAssumptionsUsedInDeterminingFairValueDerivativeLiabilityDetails" xlink:type="extended" xlink:title="41302 - Disclosure - Derivative Liability (Schedule of Assumptions Used in Determining Fair Value Derivative Liability) (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable" xlink:label="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_MeasurementInputTypeAxis" xlink:label="us-gaap_MeasurementInputTypeAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable" xlink:to="us-gaap_MeasurementInputTypeAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_MeasurementInputTypeDomain" xlink:label="us-gaap_MeasurementInputTypeDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_MeasurementInputTypeAxis" xlink:to="us-gaap_MeasurementInputTypeDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_MeasurementInputTypeDomain" xlink:label="us-gaap_MeasurementInputTypeDomain_639143609432734414"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_MeasurementInputTypeAxis" xlink:to="us-gaap_MeasurementInputTypeDomain_639143609432734414" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_MeasurementInputDebtYieldMember" xlink:label="htfl_MeasurementInputDebtYieldMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_MeasurementInputTypeDomain" xlink:to="htfl_MeasurementInputDebtYieldMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_MeasurementInputProbabilityOfBusinessCombinationOrIPOMember" xlink:label="htfl_MeasurementInputProbabilityOfBusinessCombinationOrIPOMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_MeasurementInputTypeDomain" xlink:to="htfl_MeasurementInputProbabilityOfBusinessCombinationOrIPOMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_MeasurementInputProbabilityOfChangeOfControlMember" xlink:label="htfl_MeasurementInputProbabilityOfChangeOfControlMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_MeasurementInputTypeDomain" xlink:to="htfl_MeasurementInputProbabilityOfChangeOfControlMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_MeasurementInputDiscountRateMember" xlink:label="us-gaap_MeasurementInputDiscountRateMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_MeasurementInputTypeDomain" xlink:to="us-gaap_MeasurementInputDiscountRateMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ValuationTechniqueAxis" xlink:label="us-gaap_ValuationTechniqueAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable" xlink:to="us-gaap_ValuationTechniqueAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ValuationTechniqueDomain" xlink:label="us-gaap_ValuationTechniqueDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_ValuationTechniqueAxis" xlink:to="us-gaap_ValuationTechniqueDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ValuationTechniqueDomain" xlink:label="us-gaap_ValuationTechniqueDomain_639143609432734414"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_ValuationTechniqueAxis" xlink:to="us-gaap_ValuationTechniqueDomain_639143609432734414" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_MonteCarloModelMember" xlink:label="us-gaap_MonteCarloModelMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ValuationTechniqueDomain" xlink:to="us-gaap_MonteCarloModelMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems" xlink:label="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems" xlink:to="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_DerivativeLiabilityMeasurementInput" xlink:label="us-gaap_DerivativeLiabilityMeasurementInput"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_FairValueAssetsAndLiabilitiesMeasuredOnRecurringAndNonrecurringBasisValuationTechniquesLineItems" xlink:to="us-gaap_DerivativeLiabilityMeasurementInput" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DisclosureStockBasedCompensationPlansNarrativeDetails" xlink:type="extended" xlink:title="41401 - Disclosure - Stock-Based Compensation Plans (Narrative) (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:label="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeAxis" xlink:label="srt_RangeAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="srt_RangeAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_RangeMember" xlink:label="srt_RangeMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_MinimumMember" xlink:label="srt_MinimumMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="srt_RangeMember" xlink:to="srt_MinimumMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_MaximumMember" xlink:label="srt_MaximumMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="srt_RangeMember" xlink:to="srt_MaximumMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="2" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_AwardTypeAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeStockOptionMember" xlink:label="us-gaap_EmployeeStockOptionMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_EmployeeStockOptionMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_IncentiveStockOptionsMember" xlink:label="htfl_IncentiveStockOptionsMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="htfl_IncentiveStockOptionsMember" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestrictedStockUnitsRSUMember" xlink:label="us-gaap_RestrictedStockUnitsRSUMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_RestrictedStockUnitsRSUMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeStockMember" xlink:label="us-gaap_EmployeeStockMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_EmployeeStockMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_VestingAxis" xlink:label="us-gaap_VestingAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="3" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_VestingAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_VestingDomain" xlink:label="us-gaap_VestingDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_VestingAxis" xlink:to="us-gaap_VestingDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_VestingDomain" xlink:label="us-gaap_VestingDomain_639143609432744429"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_VestingAxis" xlink:to="us-gaap_VestingDomain_639143609432744429" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_TrancheOneThroughThirtySevenMember" xlink:label="htfl_TrancheOneThroughThirtySevenMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_VestingDomain" xlink:to="htfl_TrancheOneThroughThirtySevenMember" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationAwardTrancheOneMember" xlink:label="us-gaap_ShareBasedCompensationAwardTrancheOneMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_VestingDomain" xlink:to="us-gaap_ShareBasedCompensationAwardTrancheOneMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_TrancheTwoThroughThirtySevenMember" xlink:label="htfl_TrancheTwoThroughThirtySevenMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_VestingDomain" xlink:to="htfl_TrancheTwoThroughThirtySevenMember" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_TrancheOneThroughFortyEightMember" xlink:label="htfl_TrancheOneThroughFortyEightMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_VestingDomain" xlink:to="htfl_TrancheOneThroughFortyEightMember" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PlanNameAxis" xlink:label="us-gaap_PlanNameAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="4" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_PlanNameAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_PlanNameDomain" xlink:label="us-gaap_PlanNameDomain_639143609432744429"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain_639143609432744429" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_A2025PerformanceIncentivePlanMember" xlink:label="htfl_A2025PerformanceIncentivePlanMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_PlanNameDomain" xlink:to="htfl_A2025PerformanceIncentivePlanMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_A2025EmployeeStockPurchasePlanMember" xlink:label="htfl_A2025EmployeeStockPurchasePlanMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_PlanNameDomain" xlink:to="htfl_A2025EmployeeStockPurchasePlanMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_TitleOfIndividualAxis" xlink:label="srt_TitleOfIndividualAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="5" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="srt_TitleOfIndividualAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="srt_TitleOfIndividualWithRelationshipToEntityDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="srt_TitleOfIndividualAxis" xlink:to="srt_TitleOfIndividualWithRelationshipToEntityDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/srt/2025/elts/srt-2025.xsd#srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:label="srt_TitleOfIndividualWithRelationshipToEntityDomain_639143609432754478"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="srt_TitleOfIndividualAxis" xlink:to="srt_TitleOfIndividualWithRelationshipToEntityDomain_639143609432754478" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_EmployeeWhoOwnsMoreThanTenPercentOfVotingPowerOfAllClassesOfStockMember" xlink:label="htfl_EmployeeWhoOwnsMoreThanTenPercentOfVotingPowerOfAllClassesOfStockMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="srt_TitleOfIndividualWithRelationshipToEntityDomain" xlink:to="htfl_EmployeeWhoOwnsMoreThanTenPercentOfVotingPowerOfAllClassesOfStockMember" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CommonStockCapitalSharesReservedForFutureIssuance" xlink:label="us-gaap_CommonStockCapitalSharesReservedForFutureIssuance"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_CommonStockCapitalSharesReservedForFutureIssuance" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_CommonStockCapitalSharesReservedForFutureIssuanceAnnualPercentageIncrease" xlink:label="htfl_CommonStockCapitalSharesReservedForFutureIssuanceAnnualPercentageIncrease"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="htfl_CommonStockCapitalSharesReservedForFutureIssuanceAnnualPercentageIncrease" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardPurchasePriceOfCommonStockPercent" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardPurchasePriceOfCommonStockPercent"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardPurchasePriceOfCommonStockPercent" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_ThresholdOfVotingPowerInCompanyThatTriggersIncreaseInMinimumExercisePriceOfShareBasedPaymentAwards" xlink:label="htfl_ThresholdOfVotingPowerInCompanyThatTriggersIncreaseInMinimumExercisePriceOfShareBasedPaymentAwards"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="htfl_ThresholdOfVotingPowerInCompanyThatTriggersIncreaseInMinimumExercisePriceOfShareBasedPaymentAwards" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="htfl-20260331.xsd#htfl_EmployeeStockOwnershipPlanCommonStockPurchasePriceAsPercentageOfFairMarketValue" xlink:label="htfl_EmployeeStockOwnershipPlanCommonStockPurchasePriceAsPercentageOfFairMarketValue"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="htfl_EmployeeStockOwnershipPlanCommonStockPurchasePriceAsPercentageOfFairMarketValue" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardAwardVestingRightsPercentage" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardAwardVestingRightsPercentage"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardAwardVestingRightsPercentage" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="10" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisesInPeriodTotalIntrinsicValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisesInPeriodTotalIntrinsicValue"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="11" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisesInPeriodTotalIntrinsicValue" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedStockOptions" xlink:label="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedStockOptions"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="12" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedStockOptions" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1" xlink:label="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="13" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedShareBasedAwardsOtherThanOptions" xlink:label="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedShareBasedAwardsOtherThanOptions"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="14" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedShareBasedAwardsOtherThanOptions" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SharePrice" xlink:label="us-gaap_SharePrice"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="15" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_SharePrice" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AllocatedShareBasedCompensationExpense" xlink:label="us-gaap_AllocatedShareBasedCompensationExpense"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="16" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_AllocatedShareBasedCompensationExpense" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DisclosureStockBasedCompensationPlansSummaryOfRsuActivityDetails" xlink:type="extended" xlink:title="41403 - Disclosure - Stock-Based Compensation Plans (Summary of RSU Activity) (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:label="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_AwardTypeAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestrictedStockUnitsRSUMember" xlink:label="us-gaap_RestrictedStockUnitsRSUMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_RestrictedStockUnitsRSUMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="7" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="8" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="9" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DisclosureStockBasedCompensationPlansSummaryOfEstimatedFairValueOfStockOptionsDetails" xlink:type="extended" xlink:title="41405 - Disclosure - Stock-Based Compensation Plans (Summary of Estimated Fair Value of Stock Options) (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:label="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AwardTypeAxis" xlink:label="us-gaap_AwardTypeAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_AwardTypeAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeStockOptionMember" xlink:label="us-gaap_EmployeeStockOptionMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_EmployeeStockOptionMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeStockMember" xlink:label="us-gaap_EmployeeStockMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_EmployeeStockMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRateMinimum" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRateMinimum"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRateMinimum" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRateMaximum" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRateMaximum"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRateMaximum" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate" priority="1" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="6" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DisclosureStockBasedCompensationPlansScheduleOfStockBasedCompensationExpenseDetails" xlink:type="extended" xlink:title="41406 - Disclosure - Stock-Based Compensation Plans (Schedule of Stock-Based Compensation Expense) (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:label="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeStatementLocationAxis" xlink:label="us-gaap_IncomeStatementLocationAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" xlink:to="us-gaap_IncomeStatementLocationAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeStatementLocationDomain" xlink:label="us-gaap_IncomeStatementLocationDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_IncomeStatementLocationAxis" xlink:to="us-gaap_IncomeStatementLocationDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_IncomeStatementLocationDomain" xlink:label="us-gaap_IncomeStatementLocationDomain_639143609432809751"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_IncomeStatementLocationAxis" xlink:to="us-gaap_IncomeStatementLocationDomain_639143609432809751" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_CostOfSalesMember" xlink:label="us-gaap_CostOfSalesMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_IncomeStatementLocationDomain" xlink:to="us-gaap_CostOfSalesMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ResearchAndDevelopmentExpenseMember" xlink:label="us-gaap_ResearchAndDevelopmentExpenseMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_IncomeStatementLocationDomain" xlink:to="us-gaap_ResearchAndDevelopmentExpenseMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_GeneralAndAdministrativeExpenseMember" xlink:label="us-gaap_GeneralAndAdministrativeExpenseMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_IncomeStatementLocationDomain" xlink:to="us-gaap_GeneralAndAdministrativeExpenseMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems" xlink:label="us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems" xlink:to="us-gaap_ScheduleOfEmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AllocatedShareBasedCompensationExpense" xlink:label="us-gaap_AllocatedShareBasedCompensationExpense"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_EmployeeServiceShareBasedCompensationAllocationOfRecognizedPeriodCostsLineItems" xlink:to="us-gaap_AllocatedShareBasedCompensationExpense" priority="1" use="optional"></definitionArc></link:definitionLink>
  <link:definitionLink xlink:role="http://www.heartflow.com/role/DisclosureNetLossPerShareScheduleOfOutstandingSharesOfPotentiallyDilutiveSecuritiesDetails" xlink:type="extended" xlink:title="41602 - Disclosure - Net Loss Per Share (Schedule of Outstanding Shares of Potentially Dilutive Securities) (Details)" xmlns="http://www.xbrl.org/2003/linkbase">
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:label="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable"></loc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:label="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" order="1" xlink:from="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:to="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesNameDomain" xlink:label="us-gaap_AntidilutiveSecuritiesNameDomain"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" order="1" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:to="us-gaap_AntidilutiveSecuritiesNameDomain" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesNameDomain" xlink:label="us-gaap_AntidilutiveSecuritiesNameDomain_639143609432809751"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" order="1" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:to="us-gaap_AntidilutiveSecuritiesNameDomain_639143609432809751" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RedeemableConvertiblePreferredStockMember" xlink:label="us-gaap_RedeemableConvertiblePreferredStockMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="1" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:to="us-gaap_RedeemableConvertiblePreferredStockMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeStockOptionMember" xlink:label="us-gaap_EmployeeStockOptionMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:to="us-gaap_EmployeeStockOptionMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_RestrictedStockUnitsRSUMember" xlink:label="us-gaap_RestrictedStockUnitsRSUMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="3" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:to="us-gaap_RestrictedStockUnitsRSUMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_EmployeeStockMember" xlink:label="us-gaap_EmployeeStockMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="4" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:to="us-gaap_EmployeeStockMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_WarrantMember" xlink:label="us-gaap_WarrantMember"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="5" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:to="us-gaap_WarrantMember" use="optional"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems" xlink:label="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" order="1" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems" xlink:to="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true"></definitionArc>
    <loc xlink:type="locator" xlink:href="https://xbrl.fasb.org/us-gaap/2025/elts/us-gaap-2025.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" xlink:label="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount"></loc>
    <definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" order="2" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems" xlink:to="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" priority="1" use="optional"></definitionArc></link:definitionLink></linkbase>
