Loss of Mll5 sensitizes HSCs to DNA-demethylation–induced differentiation. 5-azaCdR treatment of Mll5^−/− mice efficiently depletes HSCs from bone marrow. Mll5^+/+ or Mll5^−/− mice were injected subcutaneously with 5% DMSO (control), 5-azaCdR (1 mg/kg), or TSA (1 mg/kg) on days 1, 4, 6, 8, 11, and 13 (3 mice per genotype and drug). On day 21, bone marrow cells were harvested, retrovirally marked with YFP, and transplanted into wild-type recipients (6 mice per genotype and drug). (A) Mean and SD of percentage of treated donor-derived (YFP-positive) cells reconstituted in peripheral blood of wild-type recipients as analyzed after 4, 8 (data not shown), and 12 (data not shown) weeks by FACS analysis. (B) Mean and SD of the number of bone marrow cells in control or drug-treated mice at time of harvest (n = 3 mice per treatment per genotype). (C) Mean and SD of WBC counts in peripheral blood of treated mice at time of harvest as assessed by automated cell counting (n = 3 mice per treatment per genotype). (D) Immunophenotype of bone marrow cells in 5-azaCdR–treated mice at time of harvest. Chart shows mean and SD of cells positive for the respective surface markers (n = 3 mice per treatment per genotype). (E) Representative FACS blot (gating of viable cells) of Gr-1 and Mac-1–stained bone marrow cells of 5-azaCdR–treated mice at time of harvest. (F) Granulocyte progenitors in bone marrow of Mll5^+/+ (n = 6) or Mll5^−/− (n = 6) mice at time of harvest after intraperitoneal injection of 5-azaCdR 6 times over the course of 14 days and observation of the mice for additional 7 days. Chart shows mean and SD of number of 10-day G-CSF–responsive colony-forming cells formed per 10⁵ bone marrow cells.