IL-2 and IL-21 are closely related cytokines that might have arisen by gene duplication. Both cytokines promote the function of effector CD8⁺ T cells, but their distinct effects on antigen-driven differentiation of naive CD8⁺ T cells into effector CD8⁺ T cells are not clearly understood. We found that antigen-induced expression of Eomesodermin (Eomes) and maturation of naive CD8⁺ T cells into granzyme B- and CD44-expressing effector CD8⁺ T cells was enhanced by IL-2, but, unexpectedly, suppressed by IL-21. Furthermore, IL-21 repressed expression of IL-2Ra and inhibited IL-2–mediated acquisition of a cytolytic CD8⁺ T-cell phenotype. Despite its inhibitory effects, IL-21 did not induce anergy, but instead potently enhanced the capacity of cells to mediate tumor regression upon adoptive transfer. In contrast, IL-2 impaired the subsequent antitumor function of transferred cells. Gene expression studies revealed a distinct IL-21 program that was characterized phenotypically by increased expression of L-selectin and functionally by enhanced antitumor immunity that was not reversed by secondary in vitro stimulation with antigen and IL-2. Thus, the efficacy of CD8⁺ T cells for adoptive immunotherapy can be influenced by opposing differentiation programs conferred by IL-2 and IL-21, a finding with important implications for the development of cellular cancer therapies.