The effect of inhibition of CXCR4 signaling on progression beyond β-selection indicates a role in immunological synapse assembly. (A) Purified TCRβ⁻ DN3a cells were cocultured on OP9-DL1 cells in the presence or absence of either of the following drug combinations: Notch inhibitor (DBZ), CXCR4 inhibitor (AMD3100), a pharmacological mimic of pre-TCR downstream signaling molecule (PMA), PMA with Notch inhibitor, and PMA with CXCR4 inhibitor. Cocultures were stopped after 48 h, and PI was used to assess the effect of each treatment on survival (as shown by histograms). (B) Purified TCRβ⁻ DN3a cells were cocultured on OP9-DL1 cells in the presence or absence of either of the following drug combinations: CXCR4 inhibitor (AMD3100), a pharmacological mimic of pre-TCR downstream signaling (PMA), and PMA with CXCR4 inhibitor. Cocultures were stopped after 48 h and analyzed by flow cytometry to assess progression and differentiation (as shown by flow cytometry dot plots). The presented data are representative of three independent biological replicates with similar results.