Developmental commitment involves activation of lineage-specific genes, stabilization of a lineage-specific gene expression program, and permanent inhibition of inappropriate characteristics. To determine how these processes are coordinated in early T cell development, the expression of T and B lineage-specific genes was assessed in staged subsets of immature thymocytes. T lineage characteristics are acquired sequentially, with germ-line T cell antigen receptor-beta transcripts detected very early, followed by CD3epsilon and terminal deoxynucleotidyl transferase, then pTalpha, and finally RAG1. Only RAG1 expression coincides with commitment. Thus, much T lineage gene expression precedes commitment and does not depend on it. Early in the course of commitment to the T lineage, thymocytes lose the ability to develop into B cells. To understand how this occurs, we also examined expression of well defined B lineage-specific genes. Although lambda5 and Ig-alpha are not expressed, the mu 0 and I mu transcripts from the unrearranged IgH locus are expressed early, in distinct patterns, then repressed just before RAG1 expression. By contrast, RNA encoding the B cell receptor component Ig-beta was found to be transcribed in all immature thymocyte subpopulations and throughout most thymocyte differentiation. Ig-beta expression is down-regulated only during positive selection of CD4(+)CD8(-) cells. Thus several key participants in the B cell developmental program are expressed in non-B lineage-committed cells, and one is maintained even through commitment to an alternative lineage, and repressed only after extensive T lineage differentiation. The results show that transcriptional activation of "lymphocyte-specific" genes can occur in uncommitted precursors, and that T lineage commitment is a composite of distinct positive and negative regulatory events.