Assembly of TCRβ variable region genes is ordered during thymocyte development with Dβ to Jβ rearrangement preceding Vβ to DJβ rearrangement. The 5′ Dβ 12-RSS is required to precisely and efficiently target Vβ rearrangement beyond simply enforcing the 12/23 rule. By prohibiting direct Vβ to Jβ rearrangement, this restriction ensures Dβ gene segment use in the assembly of essentially all TCRβ variable region genes. In this study, we show that rearrangement of Vβ 23-RSSs is significantly biased to the Dβ 12-RSS over Jβ 12-RSSs on extrachromosomal recombination substrates in nonlymphoid cells that express the recombinase-activating gene-1/2 proteins. These findings demonstrate that targeting of Vβ to Dβ rearrangement can be enforced by the V(D)J recombinase in the absence of lymphoid-specific factors other than the recombinase-activating gene-1/2 proteins.