The regulation of hematopoiesis has been suggested to take place in close association with various cell types found in the bone marrow (BM) microenvironment. In the present study the role of fibroblasts, adipocytes and cell surface heparan sulfate in regulating hematopoiesis in an in vitro mouse system was examined. Mouse BM cells were allowed to adhere to a mouse embryo fibroblast cell line (C3H 10T1/2) or a clonally derived adipogenically determined derivative (Clone D) of the 10T1/2 cell line. Nonadherent cells were removed, cultures were overlaid with semisolid media supplemented with growth factors and colony formation by granulocyte-macrophage (CFU-GM), erythroid (BFU-E) and multipotential (CFU-GEMM) progenitor cells was quantitated. Adherence and co-culture of BM cells with the fibroblast cell line resulted in increased numbers of total CFU-GM and CFU-GEMM colonies. In contrast, adherence and co-culture of BM cells with the adipocytic cell line resulted in an increase only in CFU-GEMM colonies. Morphological analysis revealed a preferential adherence/growth of granulocyte and macrophage progenitors at the expense of bipotent granulocyte-macrophage progenitors to the fibroblastic cell line and an increase in the adherence/growth of granulocyte progenitors to the adipogenic cell line. Progenitor cell adherence was abolished when the fibroblastic or adipocytic cell lines were pretreated with heparitinase. These results demonstrate enhanced proliferation/differentiation of hematopoietic progenitor cells when there is direct contact between hematopoietic progenitors and cell types characteristic of those found in the microenvironment and that heparan sulfate and different types of stromal cells appear to play different roles in this interaction.