Casitas B cell lymphoma (Cbl) proteins are negative regulators for T cell antigen receptor (TCR) signaling. Their role in thymocyte development remains unclear. Here we show that simultaneous inactivation of c-Cbl and Cbl-b in thymocytes enhanced thymic negative selection and altered the ratio of CD4+ and CD8+ T cells. Strikingly, the mutant thymocytes developed into CD4+- and CD8+-lineage T cells independent of the major histocompatibility complex (MHC), indicating that the CD4+- and CD8+-lineage development programs are constitutively active in the absence of c-Cbl and Cbl-b. The mutant double-positive (DP) thymocytes exhibited spontaneous hyperactivation of nuclear factor-kappa B (NF-kB). Additionally, they failed to downregulate the pre-TCR and pre-TCR signaling. Thus, our data indicate that Cbl proteins play a critical role in establishing the MHC-dependent CD4+ and CD8+ T cell development programs. They likely do so by suppressing MHC-independent NF-kB activation, possibly through downmodulating pre-TCR signaling in DP thymocytes.