GM-CSF supernatant selectively promotes the differentiation and homeostasis of CD103⁺ DCs. Equal numbers of CFSE-labeled BM cells were plated to establish GM-DC, FL-DC, and iCD103-DC cultures. Cells were harvested after 3, 6, 9, 13, or 17 days for flow cytometric analysis. On day 9, cells were harvested, CFSE-labeled, and replated at equal numbers. (A) Kinetics for total pro-DC numbers and pre-DC numbers are shown. (B) Representative plots display Clec9A expression among pre-DCs of FL-DC and iCD103-DC cultures over time. (C) Total numbers of Clec9A⁺ pre-DCs. (D) The percentages of CD11c⁺B220^– cDCs are shown. (E) Total numbers of CD11c⁺B220^– cDCs (upper graph) and CD11c⁺B220⁺ pDCs (lower graph) over time. (F) Clec9A vs SIRPα expression among CD11c⁺B220^– cDCs is shown. (G) Absolute numbers of CD11c⁺B220^–Clec9A⁺ cDCs. (H) FL-DCs and iCD103-DCs were harvested on day 9 and labeled with CFSE before replating for 4 days with either FLT3L (FL) or FLT3L and GM-CSF (iCD103). The upper panel shows CFSE signals of CD11c⁺B220^–Clec9A⁺ cDCs, and the lower panel indicates CFSE signals of CD11c⁺B220^–SIRPα⁺ cDCs within the same cultures (black lines). Cells cultured only in presence of FLT3L on both day 0 and day 9 are overlaid as gray histograms. Representative results show 1 of 2 independent experiments (A-H).