Objective Vascular endothelial growth factor (VEGF) signaling induces Notch signaling during angiogenesis. Flt-1/VEGF receptor-1 (VEGFR-1) negatively modulates VEGF signaling. Therefore, we tested the hypothesis that disrupted Flt-1 regulation of VEGF signaling causes Notch pathway defects that contribute to dysmorphogenesis of Flt-1 mutant vessels. Approach and Results Wild-type (WT) and flt-1−/− mouse embryonic stem (ES) cell-derived vessels were exposed to pharmacological and protein-based Notch inhibitors with and without added VEGF. Vessel morphology, endothelial cell proliferation, and Notch target gene expression levels were assessed. Similar pathway manipulations were performed in developing vessels of zebrafish embryos. Notch inhibition reduced flt-1−/− ES cell-derived vessel branching dysmorphogenesis and endothelial hyper-proliferation, and rescue of flt-1−/− vessels was accompanied by a reduction of elevated Notch targets. Surprisingly, WT vessel morphogenesis and proliferation were unaffected by Notch suppression, Notch targets in WT endothelium were unchanged, and Notch suppression perturbed zebrafish intersegmental vessels (ISVs) but not caudal vein plexuses (CVPs). In contrast, exogenous VEGF caused WT ES cell-derived vessel and zebrafish ISV dysmorphogenesis that was rescued by Notch blockade. Conclusions Elevated Notch signaling downstream of perturbed VEGF signaling contributes to aberrant flt-1−/− blood vessel formation. Notch signaling may be dispensable for blood vessel formation when VEGF signaling is below a critical threshold.