Recent data have suggested that human CD34− hematopoietic stem cells (HSCs) exist, challenging the concept that HSCs necessarily and exclusively express the CD34 antigen. In mice, quiescent HSCs have been shown to be mostly CD34−, but as a consequence of 5-fluorouracil treatment or cytokine stimulation, differentiate into CD34+ cells. Of particular interest is a novel, specific marker to identify HSCs, namely the Hoechst dye efflux property, with which a distinct side population (SP) is identified. These SP cells are mostly CD34−, highly enriched for long-term repopulating cells, and durably engraft in sublethally irradiated non-obese diabetic/severe combined immunodeficient mice. Using a semiquantitative reverse transcription-polymerase chain reaction, one of the ATP-binding cassette (ABC) transporters, the breast cancer resistance protein (Bcrp) or ABC transporter G2 (ABCG2), was found to be highly expressed in SP cells as well as other primitive HSCs and to sharply drop with hematopoietic differentiation. Enforced expression of the ABCG2 cDNA resulted in a robust SP phenotype and a reduction in hematopoietic maturation. These data suggest that the Bcrp/ABCG2 gene contributes importantly to the generation of the SP phenotype, which allows for the selection of immature, pluripotent HSCs. The isolation of Bcrp/ABCG2+ cells appears to be an attractive tool to analyze and characterize HSCs, and may eventually allow for the purification of these cells for clinical purposes. In this review, current concepts on murine and human CD34− HSCs and their relationship with CD34+ HSCs are discussed.