The Runx1/Core Binding Factor- β (CBF β) transcriptional complex is required for the establishment of hematopoiesis during development. To permit the analysis of Runx1 expression in hematopoietic cell subsets, we have recently developed a novel murine line in which the expression of both full-length Runx1 and GFP is driven by the Runx1 promoter [Runx1-internal ribosomal entry site-green fluorescent protein (Runx1-GFP) knock-in mouse; Blood 103:2522]. Analysis of these mice has revealed that Runx1 is expressed in all hematopoietic lineages with the exception of erythroid cells. During our analysis, we identified in the bone marrow of these mice a cell population that expresses GFP at levels 2–5 fold higher than any other cell type (GFPhi). These cells have low forward and side scatter properties and do not express c-kit or several lineage-associated cell surface markers (lin−). In comparison to c-kit+lin− cells, these GFPhic-kit−lin− cells possess little colony forming activity in vitro. While they lack primary CFU-S activity in contrast to c-kit+lin− cells, GFPhic-kit−lin− cells possess secondary CFU-S activity. A c-kit−lin− hematopoietic stem cell (HSC) has been described by others that contributes to long-term, but not short-term, hematopoietic reconstitution in lethally irradiated recipients and may represent progenitors of c-kit+lin− HSCs in vivo (Ortiz et al. Immunity 10:173). The GFPhi bone marrow cells that we have identified share many of the properties of those c-kit−lin− cells identified by others; consequently, they likely represent the same cell population. Our ability to isolate these cells based on differential GFP expression should enable us to highly purify these GFPhic-kit−lin− cells and further characterize their immunophenotypic and biologic properties.