Hematopoietic stem cells (HSCs) are generally defined by their dual properties of pluripotency and extensive self-renewal capacity. However, a lack of experimental clarity as to what constitutes extensive self-renewal capacity coupled with an absence of methods to prospectively isolate long-term repopulating cells with defined self-renewal activities has made it difficult to identify the essential components of the self-renewal machinery and investigate their regulation. We now show that cells capable of repopulating irradiated congenic hosts for 4 months and producing clones of cells that can be serially transplanted are selectively and highly enriched in the CD150(+) subset of the EPCR(+)CD48(-)CD45(+) fraction of mouse fetal liver and adult bone marrow cells. In contrast, cells that repopulate primary hosts for the same period but show more limited self-renewal activity are enriched in the CD150(-) subset. Comparative transcriptome analyses of these 2 subsets with each other and with HSCs whose self-renewal activity has been rapidly extinguished in vitro revealed 3 new genes (VWF, Rhob, Pld3) whose elevated expression is a consistent and selective feature of the long-term repopulating cells with durable self-renewal capacity. These findings establish the identity of a phenotypically and molecularly distinct class of pluripotent hematopoietic cells with lifelong self-renewal capacity.