The effect of inhibition of Notch signaling on progression beyond β-selection indicates a role in immunological synapse assembly. (Ai) 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), a pharmacological mimic of pre-TCR downstream signaling (PMA), and PMA with Notch inhibitor. Cocultures were stopped after 48 h and analyzed by flow cytometry to assess progression and differentiation (as shown by flow cytometry dot plots). (Aii) Scatter plot represents the percentages of DN3a, DN3b, and DN4 from three independent biological replicates (each experiment is color coded [purple, blue, and orange]) in the presence of PMA, DBZ, and PMA + DBZ. Flow cytometry plots are representative of three independent biological replicates. ns, not significant; ****, P < 0.0001 (two-way ANOVA). Error bars (Aii) represent SEM.