Using 5-color fluorescence-activated cell sorting, we isolated a subset of murine pluripotent hematopoietic stem cells (PHSC) with the phenotype Lin⁻ Sca⁺ kit⁺CD38⁺ CD34⁻ that appears to fulfill the criteria for most primitive PHSC. In the presence of whole bone marrow (BM) competitor cells, these cells produced reconstitution in lethally irradiated primary, secondary, and tertiary murine transplant recipients over the long term. However, these cells alone could not produce reconstitution in lethally irradiated recipients. Rapid proliferation of these cells after BM transplantation required the assistance of another BM cell subset, which has the phenotype Lin⁻ Sca⁺ kit⁺ CD38⁻CD34⁺.