Mk budding occurs in multiple locations. (A–C) Frequency of Mks in major BM sites as quantified by flow cytometry (A), P = 0.25 one-way ANOVA; comparison of Mk frequency in the combined BM, spleen, and lungs (B), ***, P < 0.0006, one-way ANOVA with Tukey’s multiple comparisons test; and distribution of absolute Mk numbers in the BM, spleen, and lungs (C), ***, P < 0.0001; ****, P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test. Data derived from three independent mice. (D) Representative examples of large-scale z-projections showing the structure of vasculature (CD31, green) and Mk (CD41, gray) distribution in the femoral BM and spleen (n = 4 independent experiments). Scale bars, 1,000 µm. (E–G) Frequency of observed Mk morphologies within the femoral BM (o = 1,435; n = 4 independent mice), P = 0.0014, two-tailed unpaired t test with Welch’s correction (E), pelvic BM (o = 314; n = 4), P = 0.0012, two-tailed unpaired t test with Welch’s correction (F); and spleen (o = 458; n = 4), P = 0.0009, two-tailed unpaired t test with Welch’s correction (G). (H) Comparison of membrane bud (o = 176) and free Plt (o = 312) diameters in the femoral BM; P = 0.94, two-tailed unpaired t test with Welch’s correction. Cumulative, n = 4 independent experiments. (I) Frequency of observed Mk morphologies within the lungs (o = 75; n = 4). P = 0.019, two-tailed unpaired t test with Welch’s correction. Cumulative, n = 4 independent experiments. (J–M) Representative examples of F-actin distribution in budding Mks in the femur (J), pelvis (K), and spleen (L), and a proPlt-forming Mk in the lungs (M). Scale bars, 20 µm. For I–L, CD41 is shown in gray and F-actin in magenta. Membrane buds indicated with yellow arrow; proplt-forming Mk outlined in blue.