Notch signaling is required for proliferation, but not for differentiation during β-selection. (A) Experimental outline. (B) Flow cytometric analysis of NGFR⁺ gated control or TCR-β transduced human CD4⁺CD3⁻CD8α⁻CD28⁻ thymocytes after 6 days of OP9-DL1 and OP9-GFP coculture. Numbers in quadrants indicate the percentage of the corresponding population. Results shown are representative for 3 independent experiments. (C) Flow cytometric analysis of CFSE-labeled control or TCR-β–transduced CD4⁺CD3⁻CD8α⁻CD28⁻ thymocytes after 6 days of OP9-DL1 coculture. Dot plots show NGFR (marker gene for control and TCR-β transduction) expression versus CFSE label for control (top) and TCR-β (bottom) transduced cells. Numbers to the right of dot plots show ratio of CFSE^low versus CFSE^high labeled cells for transduced and untransduced cells for both sets of transduction, indicating enhanced proliferation after TCR-β transduction. (D) Flow cytometric analysis of CFSE labeled TCR-β transduced CD4⁺CD28⁻ thymocytes after OP9-DL1 (solid line) and OP9-GFP (dotted line) stromal coculture shows Notch-DL1 dependent optimal proliferation in conjunction with pre-TCR signaling.