Pathogenic variants in
NOTCH1
have been implicated in multiple types of congenital heart defects, such as bicuspid aortic valve, Tetralogy of Fallot, and hypoplastic left heart syndrome (HLHS). However, the mechanisms by which
NOTCH1
pathogenic variants cause abnormalities in human embryonic heart development are largely unknown. Here, we used CRISPR/Cas9-mediated genome editing to genetically delete
NOTCH1
in human induced pluripotent stem cells (iPSCs). We found that
NOTCH1
was dispensable for mesodermal and vascular endothelial differentiation of human iPSCs. Disruption of NOTCH activity promoted venous-specific gene expression but suppressed arterial-specific gene expression in iPSC-derived endothelial cells (iPSC-ECs). Intriguingly,
NOTCH1
deletion significantly impaired the cardiac differentiation efficiency. In
NOTCH1
homozygous knockout (
NOTCH1
-/-
) iPSC-derived cardiomyocytes (iPSC-CMs), atrial-specific genes (
NR2F2, KCNJ3
, and
MYL7
) were upregulated whereas ventricular-specific genes (
MYL2, IRX4
, and
MYH7
) were downregulated. Electrophysiological analysis by patch clamp and optical mapping indicated that atrial-like cardiomyocytes were dominant whereas the percentage of ventricular-like iPSC-CMs was dramatically reduced (<1%) in
NOTCH1
-/-
iPSC-CMs. In addition, mitochondrial respiration was reduced in NOTCH1 deficient iPSC-CMs compared to wild-type controls, which was likely attributed to the reduction of ventricular cardiomyocytes in
NOTCH1
-/-
iPSC-CMs. As
NOTCH1
is primarily expressed in endothelial cells rather than cardiomyocytes, we conclude that
NOTCH1
affects ventricular cardiomyocyte lineage commitment possibly through controlling cell fate determination of cardiac progenitors during human iPSC differentiation. Our study may provide novel insights into the mechanisms by which
NOTCH1
mutations lead to left ventricular hypoplasia in HLHS patients.