The mechanisms underlying the aging of the hematopoietic stem cell (HSC) compartment remain poorly understood. The ATP-binding cassette cell surface transport protein, ABCG2, has been identified as the transporter responsible for Hoechst 33342 (Hst) efflux in primitive stem cells and its expression is associated with the side population (SP) phenotype in both murine and human bone marrow (BM). ABCG2 expression and Hst efflux activity is highest in those cells with the greatest repopulating potential and is progressively downregulated during differentiation. The substrate profile of ABCG2, which includes a number of antineoplastic drugs, protoporphyrin IX and the chlorophyll breakdown product pheophorbide, suggest that ABCG2 transporters may function to protect stem cells from cytotoxic insults, a function which may be of great importance in stem cell maintenance. Amongst laboratory mice, the C57BL/6 strain is the longest lived and appears to accumulate HSC’s with age as assessed by both phenotype and colony forming assays. While the phenotypic features of the SP profile have been well characterized in both humans and young mice, little is known of the Hst efflux dynamics or phenotype of the SP profile in old and very old C57BL/6 mice. In order to further characterize the SP profile in old mice, whole BM was extracted from the femurs, tibiae, pelvis and thoracolumbar vertebral bodies of young (9–13 week) and old (95–108 week) C57BL/6 (Ly5.1) mice. Cells were stained with 5μg/ml Hst followed by staining with a combination of CD45.1 FITC, Sca1 PE, c-kit APC, CD34 FITC, biotinylated CD34 and lineage markers and strep PE-Texas Red. In addition, serial sampling of Hst incubated cells was performed to assess Hst efflux activity at 20 mins incubation through to 100mins. Six-color flow analysis was performed on a FACS Vantage™ (BD) cytometer and data analyzed using FlowJo™ software. Results show a marked increase in cells with an SP phenotype in old vs young mice (mean±SD 1.85%±0.88 vs 0.15%±0.09) which were more highly enriched for CD34-Sca1+ckit+ (22.2%±8.65 vs 8.89%±6.7) cells. Subdividing the SP profile into four regions (R1 to R4) from highest to lowest Hst efflux activity revealed that in old mice, SP cells with the highest Hst efflux activity were almost exclusively of a CD34-Sca1+ckit+ phenotype (82.3%±14.0 vs 11.5%±7.8), with a decreasing proportion of these cells represented throughout the remaining SP tail, though a significant proportion of cells within R4 remain CD34-Sca1+ckit+ (15.3%±7.83 vs 4.19±3.01). Similar patterns have been observed in both whole and lineage depleted BM. In addition, BM cells from old C57BL/6 mice show prolonged Hst efflux activity with an increase in cells in the SP gate at 100 mins (1.51%±0.50 vs 0.10%±0.06). We conclude that in old C57Bl/6 mice, cells accumulate which have the capacity to efflux Hst in agreement with previous reports of an increase in HSC number with age in this mouse strain.