Abstract Recent evidence suggests that mutations in the Gata1 gene may alter the proliferation/differentiation potential of hemopoietic progenitors. We had previously described that hematopoietic tissues from mice carrying the hypomorphic Gata1low mutation contain high numbers (~10%) of “unique” progenitor cells that generate colonies composed by erythroblasts, megakaryocytes and mast cells (Migliaccio et al, J Exp Med197:281, 2003). Predicted by the stochastic model of hematopoietic commitment, such a tri-lineage progenitor cell has not been isolated prospectively as yet from the marrow of normal mice. The aim of this study was to clarify the role of Gata1 in hematopoietic commitment by identifying the antigenic profile and proliferation potential of the Gata1low progenitors giving rise to tri-lineage colonies. First, we compared the frequency of granulo-monocytic (GMP), megakaryocytic-erythroid (MEP), common myeloid (CMP) and mast-cell restricted (MCP) progenitors in marrow, and spleen, from wild-type and Gata1low littermates. Next, the different populations were isolated, and their differentiation and proliferation potential characterized under conditions of limiting dilution followed by single cell re-cloning. In Gata1low mice, the frequency of cells with the antigenic profile of CMP, MEP, and GMP was normal in marrow and markedly increased in spleen, while cells with the MCP profile were not detectable. However, mutant cells isolated according to the MEP phenotype, had the “unique” property to generate mast cells and their precursors in 7 days of culture, in addition to erythroblasts and megakaryocytes. Furthermore, the progeny of ~11% of mutant MEP could be propagated in culture, as single cells, with 95% efficiency, for up of 4 months. In comparison, the progeny of wild-type MEP became extinguished in 7–14 days. In agreement with these results, tri-lineage (erythroid, megakaryocytic and mastocytic) cell lines were consistently isolated from bone marrow and spleen cells of Gata1low mice. These results indicate that cells with the phenotype corresponding to myeloid, but not those corresponding to mastocytic, progenitors are detectable in tissues from Gata1low mice. However, in these mice, the mast cell generating activity is abnormally acquired by MEP, that, therefore, are similar in phenotype, but not in function, to wild-type MEP. These results confirm the crucial role played by Gata1 in hematopoietic commitment and identify, as a new target for the Gata1 action, the restriction point at which CMP became either MEP or MCP.