The Notch signaling pathway is an evolutionarily conserved signaling mechanism and mutations in its components disrupt cell fate specification and embryonic development in many organisms. To analyze the in vivo role of the Notch3 gene in mice, we created a deletion allele by gene targeting. Embryos homozygous for this mutation developed normally and homozygous mutant adults were viable and fertile. We also examined whether we could detect genetic interactions during early embryogenesis between the Notch3 mutation and a targeted mutation of the Notch1 gene. Double homozygous mutant embryos exhibited defects normally observed in Notch1‐deficient embryos, but we detected no obvious synergistic effects in the double mutants. These data demonstrate that the Notch3 gene is not essential for embryonic development or fertility in mice, and does not have a redundant function with the Notch1 gene during early embryogenesis. genesis 37:139–143, 2003. © 2003 Wiley‐Liss, Inc.