Differentiation potential of IRF8⁺ LMPPs in vivo. Flow cytometric analysis of splenic DC subpopulations, monocytes, and neutrophils 4 days (A), 7 days (B), or 10 days (C) after intravenous transplantation of LMPP subpopulations. A total of 1000 LMPPs (CD45.2⁺) were transplanted into irradiated Ly5.1 mice (CD45.2⁻), and donor-derived (CD45.2⁺) cells were analyzed. Absolute cell numbers (per spleen of a mouse) of the indicated progeny cells derived from transplanted LMPPs are shown in the boxplots. (A-C) Values from 3 independent experiments are shown. Representative FACS plots of cDCs (D, left) and monocytes and neutrophils (D, right) on day 7. (E) Single-cell differentiation analysis of LMPP subpopulations. LMPP subpopulations were single cell-sorted into 96-well plates and cultured with Flt3L for 7 days. Following staining, cells were analyzed by flow cytometry. A total of 192 single cells of each subpopulation were analyzed. cDC1, cDC2, and pDC differentiation potential in single LMPPs was determined from the staining patterns of cells (top). Total cell numbers of indicated DC subsets yielded in 192 wells are calculated (bottom). Data are representative of 2 independent experiments with similar results. *P < .05, **P < .01, ***P < .001 (Student t test). MHC, major histocompatibility complex.