Naive T cells differentiate into various effector T cells, including CD4⁺ helper T cell subsets and CD8⁺ cytotoxic T cells (CTL). Although cytotoxic CD4⁺ T cells (CD4⁺CTL) also develop from naive T cells, the mechanism of development is elusive. We found that a small fraction of CD4⁺ T cells that express class I–restricted T cell–associated molecule (CRTAM) upon activation possesses the characteristics of both CD4⁺ and CD8⁺ T cells. CRTAM⁺ CD4⁺ T cells secrete IFN-γ, express CTL-related genes, such as eomesodermin (Eomes), Granzyme B, and perforin, after cultivation, and exhibit cytotoxic function, suggesting that CRTAM⁺ T cells are the precursor of CD4⁺CTL. Indeed, ectopic expression of CRTAM in T cells induced the production of IFN-γ, expression of CTL-related genes, and cytotoxic activity. The induction of CD4⁺CTL and IFN-γ production requires CRTAM-mediated intracellular signaling. CRTAM⁺ T cells traffic to mucosal tissues and inflammatory sites and developed into CD4⁺CTL, which are involved in mediating protection against infection as well as inducing inflammatory response, depending on the circumstances, through IFN-γ secretion and cytotoxic activity. These results reveal that CRTAM is critical to instruct the differentiation of CD4⁺CTL through the induction of Eomes and CTL-related gene.