CD41^HiHSCs rapidly generate megakaryocytes in culture without proliferation. Single CD41^−/+/Hi HSCs from UBC-GFP mice treated with G-CSF (Figure 5A) were sorted into wells of a 96-well plate and cultured in the presence of Tpo, Epo, IL-6, IL-3, and SCF. Cells were observed daily, and cell number, cell size, and megakaryocyte emergence was quantified. (A) Proliferation of single CD41^−/+/Hi cells over 14 days in culture. (B) Representative images of sorted CD41^−/+/Hi over time. (C) CD41^−/+/Hi cell fate assayed at day 14. (D) Quantification of colony diameter at day 14. “Colony size” of CD41^Hi cells was most often the diameter of remaining single cells. (E) Quantification of the largest cell diameter visible in culture over time. The dashed line at 40 μm represents the threshold above which a cell was considered “large.” (F) Size of single CD41^−/+/Hi cells before culture. (G) Quantification of megakaryocyte emergence over time. (H) Hematoxylin and eosin staining of a megakaryocyte from a CD41⁻ cell colony. Megakaryocyte is large and contains a multilobule nucleus. (I) Live imaging of representative single CD41^Hi cell on day 5 of culture. Cell stains positive for megakaryocyte marker CD41 and has a multilobule nucleus. Data are representative of 2 to 3 independent experiments. ***P < .001 by 1-way ANOVA with Tukey’s multiple comparisons test.