During thymocyte differentiation, the T cell receptor (TCR)-beta genes are rearranged before the TCR-alpha genes. Immature CD4-8- double-negative thymocytes with a productive rearrangement of the TCR-beta locus are selected to continue maturation to the CD4+8+ double-positive stage, driven by signals through the pre-TCR. The signals through the pre-TCR can be synchronized by injection of mice with anti-CD3 epsilon monoclonal antibody. Using this approach, we demonstrated coordinated induction of a triad of responses in immature thymocytes: arrest of V to DJ rearrangement in the TCR-beta locus, transient down-regulation of rearrangement-activating gene (RAG)-1 and RAG-2 transcripts, and initiation of germ-line transcription of the TCR-alpha locus. These results suggest that the transition from TCR-beta to TCR-alpha locus rearrangement is controlled by signal transduction through the pre-TCR.