Primitive hematopoietic cells from several species are known to efflux both Hoechst 33342 and Rhodamine-123. We now show that murine hematopoietic stem cells (HSCs) defined by long-term multilineage repopulation assays efflux both dyes variably according to their developmental or activation status. In day 14.5 murine fetal liver, very few HSCs efflux Hoechst 33342 efficiently, and they are thus not detected as “side population” (SP) cells. HSCs in mouse fetal liver also fail to efflux Rhodamine-123. Both of these features are retained by most of the HSCs present until 4 weeks after birth but are reversed by 8 weeks of age or after a new HSC population is regenerated in adult mice that receive transplants with murine fetal liver cells. Activation of adult HSCs in vivo following 5-fluorouracil treatment, or in vitro with cytokines, induces variable losses in Rhodamine-123 and Hoechst 33342 efflux activities, and HSCs from mdr-1a/1b^-/- mice show a dramatic decrease in Rhodamine-123 efflux ability. Thus, the Rhodamine-123 and Hoechst 33342 efflux properties of murine HSCs fluctuate in the same fashion as a number of other HSC markers, suggesting these are regulated by a common control mechanism that operates independently of that regulating the regenerative function of HSCs. (Blood. 2004;103:4487-4495)