The role of the p38 MAPK pathway in Th1 development has been controversial, because indirect manipulations of either upstream p38 activators or modifiers of p38 activity have had variable effects on IFN-γ production in CD4+ T cells. Uncertainties regarding the specificity of pharmacologic inhibition or p38 dominant negative mutants diminish the strength of conclusions about the role of the p38α isoform in Th1 development. Also, the effects of some upstream p38 activators, such as MAPK kinase 3, on Th1 development are not as strong as the effects of other manipulations, such as the expression of a dominant negative p38 mutant. Finally, embryonic lethality has prevented a direct examination of p38α-deficient T cells. To test the requirement for p38α in Th1 differentiation, we generated Ag-specific p38α+/− and p38α−/− CD4+ T cells using RAG2−/− blastocyst complementation and retroviral expression of the DO11. 10 TCR. IFN-γ production in response to TCR signaling is normal in p38α−/− T cells cultured in Th1 conditions, implying normal Th1 development. However, p38α−/− Th1 cells have a much greater defect in IFN-γ secretion stimulated by IL-12/IL-18 compared with TCR-induced IFN-γ secretion. These results suggest that the activity of p38α in Th1 cells is relatively restricted to acting in one of two alternative pathways (ie, cytokine induced) that can induce the production of IFN-γ in differentiated Th1 cells, but that p38α is not required for the process of Th1 commitment and development itself.