GMP-Ly6C⁺ cells are the lineage-committed progeny (MPs and GPs) of oligopotent GMP-Ly6C⁻ cells (GMPs). (A-B) Ten thousand GMP-Ly6C⁻ or GMP-Ly6C⁺ cells per well (A) or 10 000 LKS⁻CD34⁺FcγR^lo CMPs per well (B) were cultured in liquid media containing 10 ng/mL IL-3 and 20 ng/mL IL-6, and cultures were analyzed by flow cytometry at the indicated time points to monitor for the presence of CMPs, GMP-Ly6C⁻, GMP-Ly6C⁺, and CD11b⁺ cells (see supplemental Figure 5 for gating strategy). Data presented are from 1 experiment that is representative of at least 3 independent experiments. (C-G) Methylcellulose cultures were performed using MethoCult GFM3434 (StemCell Technologies). For primary cultures (C-F), unsorted GMPs (LKS⁻CD34⁺FcγR^hi cells), GMP-Ly6C⁻, and GMP-Ly6C⁺ cells were isolated from mouse bone marrow and 1000 cells per well were plated in methylcellulose media. (C-D) Colonies were counted and identified 7 days later: GM, mixed granulocyte/monocyte; G, pure granulocyte; M, pure monocyte colonies. Data are presented as mean plus standard deviation of triplicate culture and are representative of 3 independent experiments. (E-F) All cells were harvested from the 7-day primary cultures, colonies were dissociated, and cells were counted to permit assessment of average colony size (total harvested cells per number of colonies) (E); c-Kit⁺ cells among the progeny were identified by flow cytometry (F). Data are presented as mean plus standard deviation of 3 independent experiments. (G) The progeny of 7-day primary cultures were plated in fresh methylcellulose (50 000 cells per well) for secondary culture, and colonies were counted after a further 7 days of culture. Data are presented as mean plus standard deviation of 3 independent experiments. Statistical significance was assessed by the Student t test (*P < .05, **P < .01). n.s., not significant.