ABCG2 (BCRP/MXR/ABCP) is an ABC transporter that effluxes Hoechst 33342 and is required for the side population (SP) phenotype of hematopoietic stem cells (HSCs). Although SP cells are enriched for HSC activity, they are not a pure stem cell population and contain a significant number of differentiated and committed cells. We hypothesized that expression of ABCG2 would provide a more specific marker for HSCs, given our prior results showing that other ABC transporters were expressed in committed SP cells. To test this hypothesis, we have generated a mouse strain in which an IRES-GFP element was inserted immediately after the stop codon of Abcg2, so that GFP and the endogenous Abcg2 gene were co-expressed from the same transcriptional regulatory elements. Immunohistochemical analysis using an anti-GFP antibody showed that Abcg2/GFP allele was strongly expressed in proximal tubule cells of kidney, in the epithelial cells of small intestine, and in vascular endothelium, recapitulating the normal expression pattern of Abcg2. Using flow cytometry to analyze GFP expression in bone marrow cells, we found that 48% of SP cells expressed GFP. In the distal tip of the SP region, previously shown to have the highest concentration of HSCs, 93% of cells expressed GFP. In gated Lin- GFP+ cells within the whole bone marrow population, 55 % of the cells expressed c-Kit and Sca1, a previously identified phenotype for HSCs. We sorted Lin- cells into GFP+ and GFP− subpopulations and determined repopulation frequencies in a limiting dilution assay. Fifteen percent of recipient mice were reconstituted with 10 Lin- GFP+ cells, and 50% mice with 60 Lin- GFP+ cells. In groups transplanted with 125 to 1000 Lin- GFP+ cells, 100% of the mice were reconstituted. In contrast, no reconstitution was seen in any mice transplanted with 60,000 Lin− GFP− cells, indicating that most if not all HSCs expressed the Abcg2/GFP allele. Secondary transplant experiments and individual lineage analysis of peripheral blood cells confirmed that the Lin- GFP+ cells were long-term HSCs. These results demonstrate that expression of Abcg2 in Lin- cells defines a highly enriched population of HSCs, and is a more specific HSC marker than the SP phenotype. We are now increasing the stringency of the GFP sorting gate and transplanting mice with single cells to determine if a pure HSC population can be isolated with this simple 2 marker system.