The Notch gene family encodes large transmembrane receptors that are components of an evolutionarily conserved intercellular signaling mechanism. To assess the role of the Notch4 gene, we generatedNotch4-deficient mice by gene targeting. Embryos homozygous for this mutation developed normally, and homozygous mutant adults were viable and fertile. However, the Notch4 mutation displayed genetic interactions with a targeted mutation of the relatedNotch1 gene. Embryos homozygous for mutations of both theNotch4 and Notch1 genes often displayed a more severe phenotype than Notch1 homozygous mutant embryos. BothNotch1 mutant and Notch1/Notch4 double mutant embryos displayed severe defects in angiogenic vascular remodeling. Analysis of the expression patterns of genes encoding ligands for Notch family receptors indicated that only the Dll4gene is expressed in a pattern consistent with that expected for a gene encoding a ligand for the Notch1 and Notch4 receptors in the early embryonic vasculature. These results reveal an essential role for the Notch signaling pathway in regulating embryonic vascular morphogenesis and remodeling, and indicate that whereas theNotch4 gene is not essential during embryonic development, theNotch4 and Notch1 genes have partially overlapping roles during embryogenesis in mice.