The TCR delta enhancer (Edelta) and TCR alpha enhancer (Ealpha) play critical roles in the temporal and lineage-specific control of V(D)J recombination and transcription at the TCR alphadelta locus, working as a developmental switch controlling a transition from TCR delta to TCR alpha activity during thymocyte development. Previous experiments using a transgenic reporter substrate revealed that substitution of the 116-bp minimal Ealpha, denoted Talpha1-Talpha2, for the entire 1.4-kb Ealpha led to a premature activation of V(D)J recombination. This suggested that binding sites outside of Talpha1-Talpha2 are critical for the strict developmental regulation of TCR alpha rearrangement. We have further analyzed Ealpha to better understand the mechanisms responsible for appropriate developmental regulation in vivo. We found that a 275-bp Ealpha fragment, denoted Talpha1-Talpha4, contains all binding sites required for proper developmental regulation in vivo. This suggests that developmentally appropriate enhancer activation results from a functional interaction between factors bound to Talpha1-Talpha2 and Talpha3-Talpha4. In support of this, EMSAs reveal the formation of a large enhanceosome complex that reflects the cooperative assembly of proteins bound to both Talpha1-Talpha2 and Talpha3-Talpha4. Our data suggest that enhanceosome assembly is critical for developmentally appropriate activation of Ealpha in vivo, and that transcription factors, Sp1 and pCREB, may play unique roles in this process.