PreGMPs express functionally relevant levels of FLT3. (A) Representative FACS profiles showing FLT3 expression on different myeloid progenitors. Lineage-negative cells were first gated as Kit⁺Sca-1⁻ cells as indicated. Further gating strategies are indicated with arrows. Histograms show FLT3 expression within indicated populations; preGMPs (Lin⁻Kit⁺Sca-1⁻CD41⁻CD150⁻FcγR⁻CD105^low), GMPs (Lin⁻Kit⁺Sca-1⁻CD41⁻CD150⁻FcγR⁺), MkP (Lin⁻Kit⁺Sca-1⁻CD41⁺), pMegE (Lin⁻Kit⁺Sca-1⁻CD41⁻CD150⁺FcγR⁻CD105^low), and CFU-E (Lin⁻Kit⁺Sca-1⁻CD41⁻CD150⁻FcγR⁻CD105^high). Gray areas indicate isotype control. Percentages in panels represent percentages of FLT3-positive cells in the investigated cell populations, and represent mean values from 8 experiments with pooled mice at 8 to 12 weeks of age. (B) Single-cell RT-PCR analysis of transcriptional expression of Flt3, Csf1r (M-csfr), Csf2r (Gm-csfr), and Csf3r (G-csfr) in preGMPs and GMPs, respectively. Only cells expressing Kit were analyzed further (> 93% of cells analyzed). ■ indicate percentage of cells coexpressing Flt3. Data are means (SD) from 2 experiments, in which 88 cells were analyzed per experiment. (C) Single preGMP and GMP cells were sorted directly into media with indicated cytokines. Clones were scored after 7 to 8 days. Data represent mean (SD) number of clones from 2 to 3 experiments, with 120 cells analyzed per experiment. (D) Single preGMP FLT3-positive and -negative cells were sorted directly into media with GM-promoting cytokines (see “Clonogenic myeloid progenitor assays”). Clones were scored after 7 to 8 days. Data represents mean (SD) number of clones from 3 experiments with 60 to 120 cells analyzed per experiment.