CD8+ cytotoxic T lymphocytes (CTLs) play a major role in defense against intracellular pathogens. During development, antigen-presenting cells secrete innate cytokines such as IL-12 and IFN-α, which drive CTL differentiation into diverse populations of effector and long-lived memory cells. Using whole transcriptome analyses, the serine/threonine protein kinase Tpl2/MAP3K8 was found to be induced by IL-12 and selectively expressed by effector memory (TEM) CTLs. Tpl2 regulates various inflammatory pathways by activating the ERK mediated MAP kinase pathway in innate immune cells such as macrophages and dendritic cells. In this study, we found that a specific small molecule Tpl2 inhibitor blocked IFN-γ and TNF-α secretion as well as cytolytic activity of human CTLs. This pathway was specific for human effector CTLs, as the Tpl2 inhibitor did not block IFN-γ and TNF-α secretion from murine effector CTLs. Further, IL-12 failed to induce expression of Tpl2 in murine CTLs, and Tpl2 deficient murine CTLs did not exhibit any functional deficiency either in vitro or in vivo in response to L. monocytogenes infection. In summary, we identified a species-specific role for Tpl2 in effector function of human CTLs, which plays a major role in adaptive immune responses to intracellular pathogens and tumors.