Allelic exclusion of Vβ-to-DJβ recombination depends on asynchronous rearrangement of alleles of the gene encoding T cell receptor β in double-negative thymocytes and feedback inhibition that is maintained in double-positive thymocytes. Feedback is thought to be enforced through downregulation of Vβ accessibility. In an attempt to override this negative regulation, we introduced the enhancer of the gene encoding T cell receptor α into the Vβ gene cluster downstream of Vβ12. In double-negative thymocytes, the introduced enhancer had no measurable effect on accessibility, but Vβ12 rearrangement was stimulated and Vβ12 allelic exclusion was partially subverted. In contrast, double-positive thymocytes showed increased Vβ transcription and accessibility, but feedback inhibition of Vβ-to-DJβ recombination remained intact. Our results indicate additional regulatory constraints on Vβ-to-DJβ recombination that operate beyond the accessibility barrier.