An interleukin 3-dependent multipotential stem cell clone, LyD9, has been shown to generate mature B lymphocytes, macrophages, and neutrophils by coculture with primary bone marrow stromal cells. We report here that coculture with the cloned stromal cell lines PA6 and ST2 can support differentiation of LyD9 cells predominantly into granulocyte/macrophage colony-stimulating factor (GM-CSF)- and granulocyte (G)-CSF-responsive cells, respectively. However, these stromal cell lines were unable to support lymphopoiesis of LyD9 cells. The GM-CSF-dependent line, L-GM, which was derived from LyD9 cells cocultured with PA6 stromal cells, could differentiate into macrophages and granulocytes in the presence of GM-CSF. The L-GM line can further differentiate predominantly into neutrophils by coculture with ST2 stromal cells. The G-CSF-dependent line, L-G, which was derived from LyD9 cells cocultured with ST2 stromal cells, differentiated into neutrophils in response to G-CSF. Although the stromal cell-supported differentiation of LyD9 cells required the direct contact between LyD9 and stromal cells, a small fraction of LyD9 cells that were pretreated with 5-azacytidine could differentiate into neutrophils and macrophages without direct contact with stromal cells. These results indicate that different stromal cell lines support lineage-selective differentiation of the LyD9 stem cell and that 5-azacytidine treatment can bypass the requirement of direct contact with stromal cells, albeit with a lower frequency.