The thymus generates T cells with diverse specificities and functions. To assess the contribution of cytokine receptors to the differentiation of T cell subsets in the thymus, we constructed conditional knockout mice in which IL-7Rα or common cytokine receptor γ chain (γ(c)) genes were deleted in thymocytes just before positive selection. We found that γ(c) expression was required to signal the differentiation of MHC class I (MHC-I)-specific thymocytes into CD8(+) cytotoxic lineage T cells and into invariant natural killer T cells but did not signal the differentiation of MHC class II (MHC-II)-specific thymocytes into CD4(+) T cells, even into regulatory Foxp3(+)CD4(+) T cells which require γ(c) signals for survival. Importantly, IL-7 and IL-15 were identified as the cytokines responsible for CD8(+) cytotoxic T cell lineage specification in vivo. Additionally, we found that small numbers of aberrant CD8(+) T cells expressing Runx3d could arise without γ(c) signaling, but these cells were developmentally arrested before expressing cytotoxic lineage genes. Thus, γ(c)-transduced cytokine signals are required for cytotoxic lineage specification in the thymus and for inducing the differentiation of MHC-I-selected thymocytes into functionally mature T cells