Abstract CD109 is a monomeric cell surface glycoprotein of 170 kD that is expressed on endothelial cells, activated but not resting T-lymphocytes, activated but not resting platelets, leukemic megakaryoblasts, and a subpopulation of bone marrow CD34 + cells. Observing an apparent association between CD109 expression and the megakaryocyte lineage (MK), we sought to determine whether CD109 was expressed on MK progenitors. In fetal bone marrow (FBM), a rich source of MK progenitors, CD109 is expressed on a mean of 11% of CD34 + cells. Fluorescence activated cell sorting (FACS) of FBM CD34 + cells into CD109 + and CD109 − fractions revealed that the CD34 +CD109 + subset contained virtually all assayable MK progenitors, including the colony-forming unit-MK (CFU-MK) and the more primitive burst-forming unit-MK (BFU-MK). The CD34 +CD109 + subset also contained all the assayable burst-forming units-erythroid (BFU-E), 90% of the colony-forming units–granulocyte/macrophage (CFU-GM), and all of the more primitive mixed lineage colony-forming units (CFU-mix). In contrast, phenotypic analysis of the CD34 +CD109 − cells in FBM, adult bone marrow (ABM) and cytokine-mobilized peripheral blood (MPB) demonstrated that this subset comprises lymphoid-committed progenitors, predominantly of the B-cell lineage. CD109 was expressed on the brightest CD34 + cells identifiable not only in FBM, but also in ABM and MPB indicating that the most primitive, candidate hematopoietic stem cells (HSC) might also be contained in the CD109 + subset. In long-term marrow cultures of FBM CD34 + cells, all assayable cobblestone area forming cell (CAFC) activity was contained within the CD109 + cell subset. Further phenotypic analysis of the CD34 +CD109 + fraction in ABM indicated that this subset included candidate HSCs that stain poorly with CD38, but express Thy-1 (CD90) and AC133 antigens, and efflux the mitochondrial dye Rhodamine 123 (Rho123). When selected CD34 + cells were sorted for CD109 expression and Rho123 staining, virtually all CAFC activity was found in the CD109 + fraction that stained most poorly with Rho123. CD34 + cells were also sorted into Thy-1 +CD109 + and Thy-1 −CD109 + fractions and virtually all the CAFC activity was found in the Thy-1 +CD109 + subset. In contrast, the Thy-1 −CD109 + fraction contained most of the short-term colony-forming cell (CFC) activity. CD109, therefore, is an antigen expressed on a subset of CD34 + cells that includes pluripotent HSCs as well as all classes of MK and myelo-erythroid progenitors. In combination with Thy-1, CD109 can be used to identify and separate myelo-erythroid and all classes of MK progenitors from candidate HSCs.