Minimal proliferation and rapid differentiation of TCRβ⁺ DN thymocytes in the absence of DL-induced Notch signaling. (A) DN4 thymocytes (5 × 10³) from Lfng Tg⁻ mice were sorted and cultured on OP9 versus OP9-DL4 monolayers. Progeny were harvested, counted, and stained with anti-CD4–PE, anti-CD8–APC, and anti-CD25–FITC to measure the number of DN (diamonds), CD8 ISP (squares), and DP (circles) progeny at each time point. (B) Contour plots display expression of CD4 versus CD8 on progeny harvested on day 2. Histograms display CD25 expression on DN (solid), CD8 ISP (dotted), and DP (dashed) progeny. (C) DN3 or DN4 thymocytes from WT mice were sorted, labeled with CFSE, and cultured on OP9 versus OP9-DL4 monolayers. Progeny were harvested after 2 days and stained with anti-CD4–APC and anti-CD8–APC. Contour plots depict CD4 + CD8 versus CFSE expression.