The deletion of COOH-terminal Src kinase (Csk), a negative regulator of Src family protein tyrosine kinases (PTKs), in immature thymocytes results in the development of alpha/beta T lineage cells in T cell receptor (TCR) beta-deficient or recombination activating gene (rag)-1-deficient mice. The function of Csk as a repressor of Lck and Fyn activity suggests activation of these PTKs is solely responsible for the phenotype observed in csk-deficient T lineage cells. We provide genetic evidence for this notion as alpha/beta T cell development is blocked in lck(-/)-fyn(-/)- csk-deficient mice. It remains unclear whether activation of Lck and Fyn in the absence of Csk uncouples alpha/beta T cell development entirely from engagement of surface-expressed receptors. We show that in mice expressing the alpha/beta TCR on csk-deficient thymocytes, positive selection is biased towards the CD4 lineage and does not require the presence of major histocompatibility complex (MHC) class I and II. Furthermore, the introduction of an MHC class I-restricted transgenic TCR into a csk-deficient background results in the development of mainly CD4 T cells carrying the transgenic TCR both in selecting and nonselecting MHC background. Thus, TCR-MHC interactions have no impact on positive selection and commitment to the CD4 lineage in the absence of Csk. However, TCR-mediated negative selection of csk-deficient, TCR transgenic cells is normal. These data suggest a differential involvement of the Csk-mediated regulation of Src family PTKs in positive and negative selection of developing thymocytes.