We studied the extent of functional linkage between CD3 sigma and p56lck in pre-TCR-dependent thymocyte development. Differentiation of DN to DP cells was examined by treatment of RAG2/CD3 sigma and RAG1/p56lck double-deficient mice with anti-CD3 epsilon antibodies. The results suggest that CD3 sigma has no specific role in this maturation step, but may be important for amplification of signaling through the pre-TCR. In contrast, p56lck is the main protein tyrosine kinase associated with signaling through the pre-TCR-CD3 complex. In DP thymocytes, the Ca2+ response to anti-CD3 epsilon was totally abolished in CD3 sigma-I-but only reduced in p56lck-I-mice, and in vivo responses to anti-CD3 epsilon differed from one another. Thus, CD3 sigma and p56lck are functionally not tightly associated and their deficiencies cause distinct developmental defects.