Deltex1, Deltex2, and Deltex4 form a family of related proteins that are the mammalian homologues of Drosophila Deltex, a known regulator of Notch signals. Deltex1 is highly induced by Notch signaling in thymocytes, and overexpression of Deltex1 in T-cell progenitors can block Notch signals, suggesting that Deltex1 may play an important role in regulating Notch signals during T-cell development. A recent report found that T cells develop normally in mice carrying a targeted deletion in the Deltex1 gene (S. Storck, F. Delbos, N. Stadler, C. Thirion-Delalande, F. Bernex, C. Verthuy, P. Ferrier, J. C. Weill, and C. A. Reynaud, Mol. Cell. Biol. 25: 1437-1445, 2005), suggesting that other Deltex homologues may compensate in Deltex1-deficient T cells. We generated mice that lack expression of both Deltex1 and Deltex2 by gene targeting and further reduced expression of Deltex4 in Deltex1/Deltex2 double-deficient T-cell progenitors using RNA interference. Using a sensitive in vitro assay, we found that Notch signaling is more potent in cells expressing lower levels of Deltex proteins. Nevertheless, we were unable to detect any significant defects in thymocyte maturation in Deltex1/Deltex2 double-knockout mice. Together these data suggest that Deltex can act as a negative regulator of Notch signals in T cells but that endogenous levels of Deltex1 and Deltex2 are not important for regulating Notch signals during thymocyte development.