The βselection checkpoint during thymocyte development is a key step when immature thymocytes at double negative 3 (DN3) stage are selected to progress to the DN4 stage and beyond. While the role for the pre-T cell receptor (pre-TCR) during βselection has been established, how other receptors contribute to βselection is not well understood. Here, we identify a previously unrecognized role for CXCR4 during βselection using mice with a conditional loss of CXCR4 expression in DN thymocytes. CXCR4 physically and functionally associates with the pre-TCR and influences βselection via at least three mechanisms: regulating steady-state localization of immature thymocytes within thymic subregions, providing survival signals, and influencing efficient thymocyte proliferation post-β selection. We also show co-localization of SDF-1α, laminin 10/11 and DN3 thymocytes at the subcortical thymic regions, where the βselection events are thought to take place. We characterize the adapter protein ShcA and ERK kinase as functionally relevant signaling molecules downstream of CXCR4/pre-TCR/integrins. These data identify CXCR4 as a novel costimulator during βselection that functions together with pre-TCR and integrin ligands to orchestrate thymic development. Taken together, these data identify a novel interplay between the pre-TCR, CXCR4 and integrin receptors in regulating the βselection checkpoint.