Ectopic PU.1 strictly requires TGF-β1 stimulation to promote LCs in serum-free medium. CD34⁺ cord-blood cells were transduced with PU.1-IRES-GFP or empty control vector and then stimulated in serum-free medium supplemented with the basic cytokine combination SCF/Flt3L/GM-CSF with or without TNFα and/or TGF-β1 as indicated. (A) FACS diagrams show representative GFP⁺ cells analyzed for Langerin versus CD1a. Numbers in each quadrant indicate the percentage of cells in that quadrant. Bar diagrams show the percentages (± SEM) of generated cells expressing the indicated marker molecules: (B) CD11b; (C) CD1a; and (D) CD40. Cells were harvested and analyzed at day 7 after culture initiation. FACS diagrams are representative of 9 experiments. Bars represent mean values (± SEM) of 9 independent experiments. The differences between values indicated were significant at ^*P < .05 and ^**P < .01 (paired, 2-tailed t test).