The failure of IRF8-deficient HSCs to differentiate into B cells is cell intrinsic. (A) Sorted HSCs from IRF8^+/+ and IRF8^−/− mice were induced to differentiate in OP9 cocultures in the presence of SCF, IL-7, and Fl3tL for 7 days. The cells were stained with antibodies recognizing B220, CD19, and CD11b and analyzed by flow cytometry. Cells were gated on 7AAD⁻ living cells. The numbers are percentages of cells falling in each gate. Data represent 1 of 3 independent experiments. (B) Sorted HSCs from each group of mice were cultured in the presence of SCF, Flt3L, IL-3, and IL-6 for 18 hours before they were infected with retroviral vectors encoding an IRF8-GFP fusion protein, an IRF8 mutant K79E-GFP fusion protein, or GFP only for 24 hours in the presence of SCF, Flt3L, IL-3, IL-6, IL-7, GM-CSF, and 4 μg/mL polybrene. GFP⁺ cells were resorted and plated onto OP9 cell layer in the presence of SCF, IL-7, and Flt3L for 7 days. The cells were analyzed by flow cytometry. The numbers are percentages of cells falling in each gate. Data represent 1 of 4 independent experiments. (C) The sorted GFP⁺ cells were cultured with OP9 cells at the same conditions as in panel B and were pulsed with BrdU for 40 minutes at day 4 and analyzed by FACS. The numbers are percentages of cells falling in each gate. Data represent 1 of 2 independent experiments.