All HSCs reside in the Kit- and Sca-1–positive fraction of cultured BM cells. (A) Ten-day cultured BM cells were stained with a cocktail of biotinylated lineage-specific antibodies, followed by streptavidin-APC, anti–Sca-1–FITC, and anti-Kit–PE. Forward scatter (FSC) and side scatter (SSC) on the left plot is used to gate on hematopoietic cells. In the middle and right plots, Lin^- (negative APC-stained) and propidium iodide–negative (PI^-) cells were gated to show surface expression of Sca-1 and Kit. Numbers in the graph are the percentages of each cell fraction. (B) Expanded HSCs in cultured BM cells are Sca-1⁺Kit⁺. After 10 days of culture of total BM cells, 1.3 × 10⁴ sorted CD45.2 Sca-1⁺Kit⁺, Sca-1⁺Kit^-, Sca-1^-Kit⁺, or Sca-1^-Kit^- cells were transplanted together with 2 × 10⁵ CD45.1 competitor cells into lethally irradiated CD45.1 mice (n = 4). Peripheral blood engraftments at 3 weeks and 4 months after transplantation are shown. Error bars indicate SEM.