Dye efflux directly correlates with self-renewal. SP cells from Sca-1–enriched bone marrow (A) were stained with anti–Mac-1^PE and anti-CD4^APC antibodies. When the SP is subdivided into the geometrically similar regions R1 to R3, the percentage of cells in R1 is lowest, as indicated. Mac-1 and CD4 expression on the gated subpopulations is shown. (B) All CD45.2 Sca-1⁺ SP cells, independent of their R gate status, were sorted on the basis of Mac-1 expression and transplanted into CD45.1 lethally irradiated recipients along with 1 × 10⁵ CD45.1 WBM cells. A representative analysis of 2 different experiments is shown from animals transplanted with 20 Mac-1^– (•) or 20 Mac-1^low (○) SP cells. Engraftment values from the 2 different populations are statistically different (P < .005), except for the 4-week time point (P < .3, t test).