Abstract The architectural protein CTCF regulates the conformation and recombination of Ag receptor loci. To study the importance of CTCF in Tcrb locus repertoire formation, we created a conditional knockout mouse line that deletes Ctcf early during thymocyte development. We observed an incomplete block in thymocyte development at the double-negative to double-positive transition, resulting in greatly lowered thymic cellularity. The Tcrb repertoire was altered with a decrease in recombination of Vβ gene segments in close proximity to a CTCF binding element, resulting in an overall repertoire that was skewed in favor of Vβ gene segments with no nearby CTCF binding element. Therefore, we show that CTCF functions to diversify the Tcrb repertoire.