Single-cell transplantation analysis revealed that the cells that had the strongest dye efflux activity ("Tip"-SP cells) and had the phenotype CD34− c-Kit+ Sca-1+ Lin− (CD34− KSL cells) exhibited very strong proliferation and multilineage differentiation capacity. Ninety-six percent of the lethally irradiated mice that received a single "Tip"-SP CD34− KSL cell showed significant donor cell engraftment for long term. These findings support the hypothesis that "Tip"-SP CD34− KSL cells represent the most primitive hematopoietic stem cells that are capable of migrating into the primary site and surviving and/or proliferating with nearly absolute efficiency. This led us to propose high marrow-seeding efficiency as a specific characteristic of primitive HSCs, in addition to their self-renewal and multipotent capacity.