The Notch3 gene, a member of the Notch gene family, is expressed in a wide variety of tissues during development. We generated and analyzed Notch3-deficient mice to assess the in vivo role of the Notch3 gene. Consistent with previous observation of Krebs et al. [Characterization of Notch3-deficient mice: normal embryonic development and absence of genetic interactions with a Notch1 mutation, Genesis 37 (3) (2003) 139–143], the Notch3^−/− mice were viable, fertile, and developed normally despite abundant expression of Notch3 in various embryonic tissues. We examined the details of Notch1, 2, and 4 expressions in the Notch3^−/− embryos compared with those in wild-type embryos. As a result, we found that a deficiency in Notch3 did not affect the expression of Notch1, 2, and 4, and that either Notch1 or Notch2, or sometimes both, was always expressed in all Notch3-expressing tissues examined. These results support the idea that other Notch genes functionally compensate for Notch3 during embryonic development. We also surveyed the adult tissues of Notch3^−/− mice and found significantly fewer thymocytes in 10-week-old mice. Therefore, the thymus might be a target tissue affected by Notch3 deficiency.