E47 null HSCs displayed hyperproliferation under steady state and after transplantation stress. (A) Flk2⁻ LSKs from WT and E47 KO littermates were sorted by flow cytometry, and the expression of p21 and β-actin was examined by quantitative reverse-transcription PCR. The data are presented as KO/WT ratios for each transcript (n = 4 independent sorts). **P < .05. (B) Bone marrow cells from WT and E47 KO littermates were stained with cell surface antibodies to resolve HSC-enriched flk2⁻ LSKs, and the number of flk2⁻ LSKs was counted (n = 11 mice). (C) Surface-stained flk2⁻ LSKs from E47 WT or KO mice were fixed and then stained with antibodies to the Ki67 proliferation antigen and DAPI for cell-cycle analysis. (Left panels) Representative flow cytometric profiles used to generate the bar graph on the right (n = 3 or 4 mice). **P < .05. (D) Lethally irradiated mice reconstituted with E47 KO or WT CD45.2 donor bone marrow cells were killed at 3 weeks after transplantation. A total of 100 μg BrdU was injected into recipient mice at a 12-hour interval for 24 hours before death. The donor-derived CD45.2 flk2⁻ LSKs were fixed and stained with antibodies to BrdU, Ki67, or DAPI for proliferation and cell-cycle analysis (n = 4 mice). **P < .05. ns indicates not significant. (E) Bone marrow from E47 KO (CD45.2) and WT (CD45.1/2) mice was cotransferred into lethally irradiated CD45.1 hosts, and cell-cycle status was examined as in panel D at 2 weeks after transplantation. (n = 6 mice). P < .05.