Runx1 is critical for the survival of B cell–specified CLP progeny. (A) End point dilution of CLPs under B-cell differentiation conditions shows that Runx1-deficient CLPs form colonies at the same efficiency as Runx1^+/+ CLPs. CLPs from either Runx1^Δ/Δ [Runx1^fl/fl-Tg(vav-Cre)] or Runx1^+/+ [Runx1^+/+-Tg(vav-Cre)] were sorted, and the indicated cell number was plated on OP9 stroma cells supplemented with cytokines. Positive wells were scored on day 7 by microscopic visualization. (B) CLP progeny from Runx1-deficient mice showed a high frequency of cell death and low proliferation rates. The number of wells exhibiting cell growth was determined at both days 7 and 12 in 2 independent limiting dilution assays. The difference in the number of positive clones is shown as the percentage of surviving clones for each genotype. To estimate the proliferation rate of CLP progeny, 200 CLPs were sorted on OP9 stroma, and the total number of viable cells was determined on day 7. Shown are the results from 4 independent wells seeded in 2 independent sorts. Runx1^+/+, solid line; Runx1^Δ/Δ, stippled line. (C) FACS analysis of CLP progeny demonstrates the predominant expression of B-cell (CD19⁺/B220⁺) but not myeloid (CD11b/Gr1) antigens. For Runx1^+/+ CLPs, cells from single wells analyzed by FACS after 1 week; for Runx1^Δ/Δ CLPs, cells from 6 positive wells were pooled before analysis. (D) FACS analysis for Ly6D expression levels on pro-B/pre-B cells (CD19⁺Lin2^negB220^med) isolated from either Runx1^fl/fl Tg(vav-Cre) or Runx1^+/+ Tg(vav-Cre) mice.