Notch signaling is active in Cbfb^rss/− cells and NK-cell development is defective. (A) Lin⁻ FL cells cultured on OP9-DL1 cells, harvested at indicated time points, and stained with antibodies as described in Figure 2A. DN1 cells (Lin⁻CD45⁺CD44⁺CD25⁻) were isolated by cell sorting (purity > 95%). The expression of individual genes was normalized to Hprt (note log scale for Dxt1) and displayed as relative to day 0 Cbfb^+/+ DN1 cells. Values are averaged from 9 samples (triplicate samples from 3 independent experiments). *indicates significant difference (P≤ .05) between Cbfb^+/+ versus Cbfb^rss/− values. (B) Flow cytometric analysis of Lin⁻ FL cells cultured on OP9-DL1 (+ Flt3L, IL-7, IL-6, IL-15) in the absence and presence of the gamma secretase inhibitor (GSI) inhibitor X for 7 days. GFP⁻CD45⁺ cells were analyzed for expression of NK1.1 (NK cells) and CD19 (B cells). n_experiments = 7. (C) Lin⁻Cbfb^+/+ 14.5 dpc FL cells transduced with either MigR1 or a retrovirus expressing the Notch1 intracellular domain (ICN) and cultured for 7 days on OP9 stromal cells in the presence of Flt3L and IL-7. CD8⁻CD19⁻Gr-1⁻ cells were gated for GFP expression in the left-hand panels, and GFP⁺ cells analyzed for CD44 and CD25 expression in the right-hand panels. (D) Lin⁻Cbfb^rss/− FL cells transduced with MigR1 or ICN, and analyzed as in panel C.