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A major goal in the study of hematopoiesis is to obtain populations of primitive stem cells, free of restricted and mature cells. We previously showed that a small population of normal bone marrow, the Thy-1loLin- cells, was highly enriched for pluripotent stem cells that repopulate lethally irradiated mice. These cells also differentiated along the B lymphocyte lineage in response to the stromal elements in Whitlock-Witte cultures. These two hematopoietic activities were entirely contained in and were enriched to similar extents in the Thy- 1loLin- population. Here we show for the first time that these two activities can be resolved functionally and phenotypically. The cells that respond to the stroma in lymphoid culture are more sensitive to the cytotoxic drug 5-Fluorouracil than are stem cells. Furthermore, we have derived a new monoclonal antibody, Fall-3, that detects primitive stem cells but does not label the B cell precursor. This indicates that the small Thy-1loLin- population is heterogeneous, containing precursors restricted to the B cell lineage as well as pluripotent stem cells. Antibody Fall-3 defines a novel stem cell antigen, expressed on all primitive stem cells and thus, will be useful in the further characterization and isolation of both stem cells and B cell precursors.