Pre-TCR and Notch signaling collaboratively induce blastogenesis and proliferation of TCRβ⁺ DN thymocytes. DN3 or DN4 thymocytes (2 × 10⁴) from WT mice were sorted onto OP9 versus OP9-DL4 monolayers and cultured for 24 hours. DN3 progeny (A) and DN4 progeny (B) were harvested and stained with anti-CD4–PE, anti-CD8–APC, anti-CD25– FITC, and anti-TCRβ CyCHR. Contour plots display expression of CD25 versus TCRβ on total progeny (left) or CD4 versus CD8 expression on TCRβ⁺ progeny (right). (C-E) DN3 progeny (left) and DN4 progeny (right) were harvested and stained with anti-CD71–FITC, CD98-PE, and anti-TCRβ CyCHR. Histograms display FSC (C), CD98 (D), and CD71 (E) profiles on TCRβ⁺ or TCRβ⁻ progeny 24 hours after culture on OP9 (dotted histograms) versus OP9-DL4 (solid histograms).