Ag receptor loci are regulated to promote allelic exclusion, but the mechanisms are not well understood. Assembly of a functional TCR β-chain gene triggers feedback inhibition of V β-to-DJ β recombination in double-positive (DP) thymocytes, which correlates with reduced V β chromatin accessibility and a locus conformational change that separates V β from DJ β gene segments. We previously generated a Tcrb allele that maintained V β accessibility but was still subject to feedback inhibition in DP thymocytes. We have now further analyzed the contributions of chromatin accessibility and locus conformation to feedback inhibition using two novel TCR alleles. We show that reduced V β accessibility and increased distance between V β and DJ β gene segments both enforce feedback inhibition in DP thymocytes. Copyright © 2011 by The American Association of Immunologists, Inc.