Abstract Stem cells from a variety of tissues can be identified by a side population (SP) phenotype based on Hoechst 33342 dye efflux. The Abcg2 transporter is expressed in hematopoietic stem cells (HSCs) and confers this dye efflux activity. To further explore the relationship among Abcg2 expression, the SP phenotype, and HSC activity, we have generated mice in which a green fluorescent protein (GFP) reporter gene was inserted into the Abcg2 locus. In these mice, the majority of bone marrow (BM) cells that expressed the Abcg2/ GFP allele were Ter119+ erythroid cells. The Abcg2/GFP allele was also expressed in approximately 10% of lineage-negative (Lin−) and in 91% of SP cells using stringent conditions for the SP assay. Flow cytometric sorting was used to isolate various Abcg2/GFP+ BM cell populations that were then tested for HSC activity in transplant assays. There was significant enrichment for HSCs in sorted Lin−/ GFP+ cells, with a calculated HSC frequency of approximately one in 75. There was no HSC activity detected in Lin−/GFP+ cells. Altogether, these results show that Abcg2 is expressed on essentially all murine BM HSCs and can be used as a prospective marker for HSC enrichment.