The development and function of alpha beta T lymphocytes depend on signals derived from pre-T and alpha beta T cell receptors (preTCR and alpha beta TCR) (reviewed in refs 1, 2). The engagement of these receptors leads to the activation of Lck and Fyn(3,4), which are protein tyrosine kinases (PTKs) of the Src family. It remains unclear to what extent the activation of Src-family PTKs can direct the differentiation steps triggered by preTCR and alpha beta TCR. Here we show that the inactivation of the negative regulator of Src-family PTKs, carboxy-terminal Src kinase (Csk)(5), in immature thymocytes abrogates the requirement for preTCR, alpha beta TCR and major histocompatibility complex (MHC) class II for the development of CD4(+)8(+) double-positive and CD4(+) single-positive thymocytes as well as peripheral CD4 alpha beta T-lineage cells. These data show that Csk and its substrates are required to establish preTCR/alpha beta TCR-mediated control over the development of alpha beta T cells.