Chemical inhibition of signaling pathways is not sufficient to block CSF-1–mediated lineage instruction. (A) GMPs were sorted as single cells into 384-well plates containing medium with indicated cytokines, 10% FCS, and different signaling inhibitors. Colonies were identified after 5 days of culture according to morphology and lineage-specific markers (F4/80 for M and Ly6G for G). Immature colonies (including small colonies) consisted of cells negative for both markers. (B) Clonogenicity (ie, capability to form colonies existing of ≥2 cells) and lineage output of cells cultured in the presence of CSF-1 and indicated inhibitors (PI3Ki = 50 μM Ly294002; MEKi = 10 μM U0126; and SFKi = 2 μM SU6656). (C) Clonogenicity and lineage output of cells cultured in the presence of SCF, IL-3 and IL-6, and indicated inhibitors (n = 5 experiments). P values are shown as follows: ***P < .001 compared with DMSO control. DMSO, dimethyl sulfoxide; FCS, fetal calf serum.