Two highly related receptor tyrosine kinases, TIE and TEK, comprise a family of endothelial cell-specific kinase. We established monoclonal antibodies against them and performed detailed analyses on their expression and function in murine hematopoietic stem cells (HSCs). TIE and TEK were expressed on 23.7% and 33.3% of lineage marker-negative, c-Kit⁺ and Sca-1⁺ (Lin⁻ c-Kit⁺ Sca-1⁺) HSCs that contain the majority of day-12 colony-forming units-spleen (CFU-S) and long-term reconstituting cells, but not committed progenitor cells. Lin⁻ c-Kit⁺ Sca-1⁺ cells were further divided by the expression of TIE and TEK. TIE⁺ and TEK⁺ HSCs as well as each negative counterpart contained high proliferative potential colony-forming cells and differentiated into lymphoid and myeloid progenies both in vitro and in vivo. However, day-12 CFU-S were enriched in TIE⁺ and TEK⁺ HSCs. Our findings define TIE and TEK as novel stem cell marker antigens that segregate day-12 CFU-S, and provide evidence of novel signaling pathways that are involved in the functional regulation of HSCs at a specific stage of differentiation, particularly of day-12 CFU-S.