It has been hypothesized that distinct stromal cells from niches within the microenvironment that selectively regulate stem cell functions. To test this hypothesis, we derived a panel of matched stromal cell lines from murine fetal liver. The lines were immortalized with a retroviral vector encoding a temperature sensitive SV40 T antigen, to provide a snapshot of potential heterogeneity of the in vivo stroma compartment. All the stromal cell lines tested, supported the proliferation and differentiation of myeloid cells in Dexter type bone marrow cultures. Furthermore, RT-PCR analysis indicates that these lines are similar with respect to the production of an array of cytokines. However, the stromal cell lines differed markedly in their ability to maintain in vitro stem cells with in vivo repopulating capacity. Stem cell levels were measured in the competitive repopulation assay, following 3 weeks of coculture on individual stromal cell lines. Three classes of stromal cell lines were identified: (1) lines that did not support stem cells, (2) lines that sustained low levels of stem cells that often showed limited persistence in vivo, and (3) an infrequent line (1 out of 16 lines tested) that maintained high levels of primitive, long-term repopulating stem cells. This suggests that stromal cells that can support primitive stem cells are rare in the hematopoietic microenvironment. Taken together, these data substantiate the hypothesis that distinct stromal cells interact selectively with stem cells.