The lck gene encodes a lymphocyte-specific protein tyrosine kinase of the nonreceptor type that is implicated in signal transduction pathways emanating from the CD4 and CD8 coreceptors. Previous studies also support a role for p56lck in regulating T cell receptor beta gene rearrangements and, more generally, thymocyte development. Here we report that a mutant form of p56lck, which is incapable of interacting with CD4 or CD8, behaves indistinguishably from association-competent p56lck with respect to its ability to affect thymocyte maturation. The effects of p56lck remained specific in that the closely related src-family kinase p59hck was incapable of substituting for p56lck in arresting beta locus gene rearrangements. These data support the view that src-family kinases perform highly specialized and often nonoverlapping functions in hematopoietic cells, and that p56lck acts independently of its association with CD4 and CD8 to regulate thymocyte development.