Hbo1 deleted HSCs exit quiescence and fail to undergo symmetric self-renewing divisions. (A-B) Hbo1^fl/+;Mx1Cre and Hbo1^fl/fl;Mx1Cre mice (n = 5 animals per genotype) were injected 3× with poly(I:C) (2.5 mg/kg BW per dose, doses 48 hours apart). Bone marrow was collected 7 days after the first poly(I:C) injection. Representative FACS plots and percentages of HSC^SLAM at different stages of the cell cycle. Note the more than twofold increase in cells in S phase. (C-D) Hbo1^fl/+;CreER and Hbo1^fl/fl;CreER mice (n = 6 animals per genotype) were treated with tamoxifen (150 mg/kg BW per dose, doses 48 hours apart). Bone marrow was collected 7 days after the first tamoxifen dose. Representative FACS plots and percentages of HSC^SLAM at different stages of the cell cycle. (E-F) Hbo1^fl/+;CreER and Hbo1^fl/fl;CreER mice (n = 3 animals per genotype) were treated 3× with tamoxifen (150 mg/kg BW per dose, doses 24 hours apart). LSKCD150⁺ cells were sorted by FACS 3 days after the first tamoxifen treatment and plated on an 8-well chamber slide coated with GFP-labeled OP9 feeder cells in StemSpan medium with SCF (30 ng/mL) and FLT3 ligand (30 ng/mL) overnight. Nocodazole (20 nM)  was added after ∼16 hours. Cells were cultured for another 24 hours and then fixed and stained with antibodies to numb and GFP and counterstained with DAPI. (E) Representative of images of symmetric self-renewing (SS), asymmetric (AS), and symmetric differentiating divisions (SD) with numb (red) and DAPI (blue; GFP green, not shown). (F) Enumeration of cells with division patterns of SS, AS, or SD division in LSKCD150⁺ cells. (G) Model proposing HSCs lacking HBO1 exited the quiescent state through SD instead of SS division and consequently declined in numbers. Data are displayed as mean ± SEM and were analyzed using a 2-tailed Student t test.