While it has been shown in several publications that the serine-threonine kinase PKCθ is required for efficient activation of mature T lymphocytes, the role of PKCθ in T cell development in the thymus is somewhat controversial. In this study, using knockout mice, we show that PKCθ is important in positive selection. The thymus of PKCθ−/− animals contains significantly less mature single positive T cells compared to wild-type controls. Biochemically, PKCθ deficient thymocytes show defective activation of the transcription factors AP-1, NFAT and NFκB as well as impaired phosphorylation of the MAP kinase ERK after T cell receptor stimulation in vitro. Together, these results reveal a crucial role of PKCθ in positive selection of thymocytes in a pathway leading to the activation of ERK, AP-1, NFAT, and NFκB.