HSCs are contained within the CD48^– fraction while colony-forming progenitors are almost entirely in the CD48⁺ fraction of fetal liver cells. (A) Twelve percent of fetal liver cells expressed CD48. (B) CD48 expression was heterogeneous in the Thy^lowSca-1⁺lineage^–Mac-1⁺ HSC population, with 51% of cells being CD48⁺. For this analysis CD48, Thy-1, Sca-1, lineage marker, and Mac-1 staining were detected in the FITC, PE-Cy5, APC-Cy7 (PR), PE, and APC channels, respectively. (C) Recipient mice received transplants of 5750 CD48⁺ fetal liver cells (blue lines) or 44 250 CD48^– fetal liver cells (red lines). Cell doses were based on the proportion of CD48⁺ versus CD48^– cells in 50 000 fetal liver cells (which gives long-term multilineage reconstitution in nearly all recipient mice), as done in previous studies of marker expression by HSCs.6,14,24,26 Each line represents the frequency of donor-derived myeloid, B, or T cells in a single mouse. The black line at 0.3% represents the background threshold, meaning that reconstitution could not be detected in mice falling below this line. All long-term multilineage reconstituting activity was contained within the CD48^– cell fraction (7 of 7 mice). CD48⁺ cells gave transient multilineage reconstitution (4 of 9 mice) or B-lineage-only reconstitution (5 of 9 mice). Data are from 1 of 2 independent experiments that gave similar results. (D) Fetal liver cells were divided based on their expression of all possible combinations of the CD48, CD244, and CD150 SLAM family receptors and sorted into methylcellulose to measure colony-forming activity. Each bar represents the percentage of all colony-forming progenitors of each type that fell within the indicated cell fraction. For example, around 80% of all CFU-GM, CFU-G, and CFU-M in the fetal liver fell within the CD48⁺CD244^–CD150^– cell population. Error bars indicate SD.