STAT5B, but not STAT5A, has an important role in HSC dormancy. (A-B) HSC flow cytometry analyses of wt, Stat5a^−/−, or Stat5b^−/− BM. (A) Representative FACS plots showing HSC/multipotent progenitor (MPP)1 (CD150⁺CD48⁻), MPP2 (CD150⁺CD48⁺), MPP3/4 (CD150⁻CD48⁺), and MPP5 (CD150⁻CD48⁻) cell populations gated on LSK cells. (B) Relative quantification of HSC, MPP1-5, and LSK cells (n ≥ 6; mean ± standard error of the mean [SEM]). HSC subpopulations: HSC (LSK, CD34⁻CD48⁻CD150⁺CD135⁻), MPP1 (LSK, CD34⁺CD48⁻CD150⁺CD135⁻), MPP2 (LSK, CD34⁺CD48⁺CD150⁺CD135⁻), MPP3 (LSK, CD34⁺CD48⁺CD150⁻CD135⁻), and MPP4 (LSK, CD34⁺CD48⁺CD150⁻CD135⁺). (C-F) Single cell RNA-Seq of FACS-sorted LSK cells of WT, Stat5a^−/−, or Stat5b^−/− BM (n = 3 pooled/genotype). (C) Experimental workflow. (D) Force-directed graphs of WT, Stat5a^−/−, or Stat5b^−/− LSKs, color code as shown in supplemental Figure 1C. (E) Changes in HSC_1 and HSC_2 cluster sizes of Stat5a^−/− or Stat5b^−/− relative to WT. (F) Absolute differences in the percentage of cells expressing known genes associated with dormant HSCs^39,41-43 in pooled clusters HSC_1 and HSC_2 of Stat5a^−/− or Stat5b^−/− compared with WT. (G-H) HSC cell cycle analyses of WT, Stat5a^−/−, or Stat5b^−/− BM. (G) Representative FACS plots (KI67/DAPI) of HSC/MPP1 with gating strategy and indicated percentages, and (H) quantification of cell cycle phase distributions of HSC/MPP1 cells (n = 5; mean ± SEM). Levels of significance were calculated using 1-way analysis of variance (ANOVA) (B,H). *P < .05; **P < .01; ***P < .001.