Abstract Body (Do not enter title and authors here): Congenital heart disease (CHD) affects ~1% of live births, and although both genetic and environmental etiologic contributors have been identified, the majority of CHD lacks a definitive cause, suggesting the role of gene-environment interactions (GxE) in disease pathogenesis. Maternal diabetes mellitus (matDM) is among the most prevalent environmental risk factors for CHD. However, there is a substantial knowledge gap in understanding how matDM acts upon susceptible genetic backgrounds to increase disease expressivity. Previously, we reported a novel GxE between Notch1 haploinsufficiency and matDM leading to increased penetrance of CHD. Here, we demonstrate a cell lineage specific effect of Notch1 haploinsufficiency in matDM-exposed embryos, implicating endothelial/endocardial derived tissues in the developing heart, resulting in impaired atrioventricular cushion morphogenesis. Further, we find that NOTCH1 haploinsufficient endothelial cells have an altered transcriptomic response to oxidative stress compared to control endothelial cells, with an exacerbated effect on gene regulatory networks critical for endocardial cushion morphogenesis. In support of this, mitigation of matDM-associated oxidative stress via SOD1 overexpression did not rescue CHD in Notch1 haploinsufficient mice as compared to wildtype littermates. Our results demonstrate the combinatorial interaction of matDM-associated oxidative stress and a genetic predisposition, Notch1 haploinsufficiency, on endocardial cushion development, supporting a GxE model for CHD etiology and suggest that antioxidant strategies maybe ineffective in genetically-susceptible individuals.