Enforced c-Src activity is sufficient to drive CSF-1–independent M differentiation of GMPs at the expense of granulocytic fate. (A) preGMPs were freshly isolated, and cultured in 100 ng/mL SCF and Flt3L while being transduced with lentiviruses encoding variants of signaling molecules CA or DN. After 3 days, transduced ivGMPs were either sorted and cultured in permissive colony assay medium (M3434, containing SCF, IL-3, IL-6, and EPO), or sorted as single cells into 384-well plates containing SCF, IL-3, IL-6, and 10% FCS. After 5 to 7 days in culture, colonies were enumerated and identified using lineage-specific markers (F4/80 for M and Ly6G for G). Immature colonies consisted of cells negative for both markers. (B) Lineage output in methylcellulose colony assay (clonogenicity VENUS = 49% ± 7.2% SD, Akt CA = 33% ± 7.4% SD, Akt DN = 39% ± 8.4% SD, c-Src CA = 35% ± 9.2%, c-Src DN = 51% ± 7.4% SD, Fyn CA = 45% ± 9.4% SD, Lyn CA = 52% ± 6.4% SD, and Hck CA = 46% ± 7.3% SD; n = 3 to 6 experiments). (C) Clonogenicity and lineage output of single cells cultured in 384-well plates (n = 4 to 5 experiments). P values are shown as follows: **P < .01; ***P < .001 compared with VENUS control (only calculated for M). CA, constitutive active; DN, dominant negative.