The physiological roles of PKCα and PKCθ were defined in T cell immune functions downstream of the antigen receptor. To investigate the hypothesis that both PKC isotypes may have overlapping functions, we generated mice lacking both genes. We find that PKCα−/−/θ−/− animals have additive T cell response defects in comparison to animals carrying single mutations in these genes. Our studies demonstrate that the activities of PKCα and PKCθ converge to regulate both IL-2 cytokine responses and T cell intrinsic alloreactivity in vivo. Mechanistically, this PKCα/θ crosstalk primarily affects the NFAT transactivation pathway in T lymphocytes, as observed by decreased phosphorylation of Ser-9 on GSK3β, reduced nuclear translocation and DNA binding of NFAT in isolated PKCα−/−/θ−/− CD3+ T cells. This additive defect proved to be of physiological relevance, because PKCα−/−/θ−/− mice demonstrated significantly prolonged allograft survival in heart transplantation experiments, whereas both PKCα−/− and PKCθ−/− mice showed only minimal graft prolongation when compared to wild type controls. While PKCθ appears to be the rate-limiting PKC isotype mediating T lymphocyte activation, we here provide genetic evidence that PKCα and PKCθ have overlapping functions in alloimmunoreactivity in vivo and both PKCθ and PKCα isotypes must be targeted to prevent organ allograft rejection.