While a complete knockout of NOTCH1 has not been observed clinically, this study shows an extreme example of roles of NOTCH1 in cardiac cell proliferation and lineage determination by maintaining the balance of cardiac progenitor cell populations and the proper specification of cardiomyocyte subtypes, which may be dysregulated in NOTCH1-associated HLHS. Here, NOTCH1 is linked to hypoplastic left heart syndrome.