Abstract: To study age‐related changes of mouse bone marrow (BM) cells and hematopoietic stem cells (HSCs), we isolated rhodamine‐123^low (Rh^low) Thy1.1^low Lin⁻Sca‐1⁺ (TLS) HSCs from the BM of old mice and compared their functional characteristics to cells of the same phenotype isolated from young mice. We observed impaired recovery of B lymphocytes and decreased self‐renewal in recipients of old Rh^low cells compared to young Rh^low cells. Blockade of Rh efflux using verapamil improved lymphoid reconstitution by enriched HSCs, and isolation of aged HSCs based on efflux of a fluorescent multi‐drug resistance (MDR) substrate (Bodipy‐verapamil) resulted in enrichment of HSC activity equivalent to that obtained with Rh. These observations suggest a complex relationship between MDR activity and HSC function during aging. To address whether the difference between young and aged donors was intrinsic to the HSC compartment or was due to a shift in HSC phenotype, we co‐transplanted normal BM derived from young or old donors and followed repopulation simultaneously in the same recipient animals. In a parallel experiment, we co‐transplanted HSCs purified from old donors with BM derived from young donors. In both experiments, transplants were given to both young and old recipients. The results show a clear defect in B‐cell engraftment from either BM or HSCs of old donors, irrespective of the age of the recipient. In contrast, myeloid engraftment was predominantly derived from BM or HSCs derived from aged donors, again irrespective of recipient age. These data suggest a stem cell basis for B‐cell immuno‐senescence and the increased incidence of myelocytic leukemia in elderly people.