(A) Colony formation by single HSCs from control or E2 treated Esr1fl/fl (WT) and Mx1-Cre; Esr1fl/fl (Esr1Δ/Δ) mice (96 wells per animal, n = 4 assays/group). Colonies were collected, cytospun, and scored after Wright Giemsa staining. gemM: granulocyte, erythroid, monocyte, megakaryocyte; gmM: granulocyte monocyte megakaryocyte; gme: granulocyte, monocyte, erythroid; gm: granulocyte, monocyte; M: megakaryocyte; m: monocyte; e: erythroid. Red and green lines indicate significant interaction of treatment and genotype of gemM and gm, respectively (***p<0.001, ANOVA) (B) Megakaryocyte differentiation potential in collagen-based MegaCult assays (n = 6, three independent experiments, two technical replicates per experiment). Meg, colonies containing exclusively megakaryocytes as indicated by cholinesterase staining; Mixed, colonies containing both megakaryocytes and other myeloid cells; Non, colony with no megakaryocytes. *p<0.05, ANOVA. (C) Numbers of CD41+ megakaryocytes as indicated by immunofluorescent staining of bone marrow sections (n = 10, 5 fields of view per section). (D) Representative images of CD41 stained bone marrow sections from oil- and E2-treated mice. Scale bar represents 50 μm. (E–J) Levels of donor (GFP+) engraftment in recipient mice that were transplanted with 100 GFP+ HSCs (oil- or E2-treated Ubc-GFP; Esr1fl/fl (n = 4 donors each, 24 and 26 recipients respectively) or Ubc-GFP; Mx1-Cre; Esr1fl/fl (n = 3 donors each, 14 and 23 recipients respectively) and 2 × 105 competitor GFP- cells. Each panel represents donor chimerisms in (E) overall mononuclear cells, (F) Mac-1/Gr-1+ myeloid cells, (G) CD41+ FSC/SSClow platelets, (H) Ter119+ FSC/SSClow red blood cells, (I) B220+ B-cells, and (J) CD3+ T-cells. All data represent mean ±standard deviation; *p<0.05; **p<0.01; and ***p<0.001 by Student’s t-test unless otherwise noted.