<jats:title>Abstract</jats:title> <jats:p>Monoallelic expression of antigen receptor (AgR) genes is assumed to be critical for the proper selection and function of T and B lymphocytes. V(D)J recombination of Tcrb, Igh, and Igk loci is regulated such that most cells assemble and express functional AgR genes from one allele (allelic exclusion). The V gene segment recombination signal sequences (RSSs) of Tcrb and Igh loci are weak and have been proposed to mediate monoallelic V-to-DJ recombination. To test the role of Vβ RSSs in promoting Tcrb allelic exclusion, we created and studied mice harboring replacements of the DJCβ-distal V2 and -proximal V31 RSSs with the stronger 3′Dβ1 RSS. Here, we show a substantial role for weak Vβ RSSs in limiting: Vβ recombination frequency, biallelic functional Vβ-to-DJβ recombination and biallelic TCRβ expression, and dual TCRβ chain expression encoded by a single Tcrb allele. Our data indicate that weak Vβ RSSs limit Vβ recombination to promote monogenic Tcrb assembly in the time window before feedback inhibition halts Vβ recombination. Given that AgR allelic exclusion is most stringently applied to Tcrb and Igh, whose assembly and expression drives cellular proliferation, we hypothesized that mechanisms directing monoallelic induction of RAG double strand breaks (DSBs) evolved at Tcrb and Igh loci in part to suppress DSBs from entering S phase and forming oncogenic AgR translocations. We find that p53D/D mice with V2 and V31 RSS replacements succumb more rapidly to thymic lymphomas than their p53D/D counterparts, which is consistent with our model. As Vβ RSS replacement mice exhibit dramatic shifts in the Vβ repertoire, we also have begun using them to test relationships between Vβ repertoire and host defense.</jats:p>