Casp9^−/− HSCs are functionally impaired. (A) FLCs from Casp9^−/− or Casp9^+/+ pups (Ly-5.2⁺) were transplanted 1:1 in competition with WT congenic (Ly-5.1⁺) FLCs into lethally-irradiated WT congenic (Ly-5.1⁺Ly-5.2⁺) recipients. Peripheral blood was analyzed for donor chimerism at monthly intervals. Casp9^−/− cells demonstrated a competitive disadvantage at 1 to 5 months, suggesting that their short-term repopulating cells are functionally impaired. Each curve represents the mean ± SEM of 3 to 4 recipients from separate FLC donors (n = 3 donors per genotype, 21 recipients total). The dotted line indicates no difference in competitive advantage. (B) In secondary transplants, bone marrow cells obtained from primary recipients of Casp9^−/− donor cells failed to reconstitute the hematopoietic compartment of lethally-irradiated secondary recipients, although secondary transplants from donors engrafted with Casp9^+/+ cells were successful. Each curve represents the mean ± SEM of 4 recipients from separate donors (n = 3 donors of each genotype, 24 recipients total). (C) Noncompetitive transplants with Casp9^−/− or Casp9^+/+ FLCs had similar engraftment in lethally irradiated WT congenic (Ly-5.1⁺) recipients (mean % donor cells at 1 month of 87.0% and 81.2% for Casp9^+/+ and Casp9^−/−, respectively; P = .086). Each curve represents the mean ± SEM of 1 to 5 recipients from Casp9^−/− (n = 7) or Casp9^+/+ (n = 5) donors. (D) Recipients of Casp9^−/− FLCs developed leukopenia, anemia, and B-cell lymphopenia compared with recipients of Casp9^+/+ FLCs. ***P < .0005.