(A) WT and VPS33B-null BM CD45.2 cells along with CD45.1 competitor cells were injected into lethally irradiated CD45.1 recipient mice (n = 5; **P < 0.01, ***P < 0.001 using Student’s t test). Repopulation was analyzed at 3, 8, and 16 weeks after transplantation. (B) Multilineage contribution of donor cells in the primary recipients at 16 weeks post-transplantation (n = 5; *P < 0.05 using Student’s t test). (C) Secondary transplantation was performed with FACS-purified donor CD45.2 BM cells from WT and VPS33B-null primary recipients. Repopulation was analyzed at 4, 8, and 16 weeks after transplantation (n = 5; **P < 0.01, ***P < 0.001 using Student’s t test). (D) Multilineage contribution of donor cells in secondary recipients 16 weeks after transplantation (n = 5; *P < 0.05 using Student’s t test). (E) Vps33b^wt/wt Scl-Cre-ER (VPS33B^+/+) and Vps33b^fl/fl Scl-Cre-ER (VPS33B^–/–) mice were intraperitoneally administered 150 mg/kg 5-FU weekly for 3 times (arrows), and the survival rates were analyzed (n = 7; *P < 0.05 using log-rank test). (F) Vps33b^fl/fl Scl-Cre-ER⁺ BM cells or Vps33b^wt/wt Scl-Cre-ER⁺ BM cells (3 × 10⁵ cells) along with competitor cells were transplanted into lethally irradiated CD45.1 recipient mice, followed by the treatment with tamoxifen 8 weeks after transplantation and analysis for repopulation from 2 to 24 weeks after treatment. Vps33b deletion (arrow) was shown after 2-week treatment (n = 5; **P < 0.01, ***P < 0.001 using Student’s t test). (G) Multilineage contribution of donor cells 24 weeks after transplantation (n = 5; *P < 0.05 using Student’s t test). (H and I) WT and VPS33B-null LT-HSCs were analyzed for cell cycle stage by staining with Hoechst33342/Pyronin Y (H), and the frequencies of the G0, G1, and S-G2-M fractions were quantified (n = 3; *P < 0.05, ***P < 0.001 using Student’s t test) (I). (J) Apoptosis was measured in LT-HSCs from VPS33B^+/+ and VPS33B^–/– mice by using annexin V/7-AAD staining (n = 5; ***P < 0.001 using Student’s t test). (K) Representative images of LT-HSCs from VPS33B^+/+ and VPS33B^–/– mice 10 days after culturing in basic medium (SCF+TPO) (n = 6). (L–N) Cell numbers (L), percentages of LSK cells (M), and percentages of apoptotic cells (N) were evaluated in cultured WT and VPS33B-null LT-HSCs (n = 6; *P < 0.05, **P < 0.01, ***P < 0.001 using Student’s t test). Experiments were conducted 3 to 5 times for validation.