We dissected the functions of the microenvironment of bone marrow (BM) and fetal liver (FL) at the cellular level by cloning individual stromal calls and characterizing their phenotypical and functional features. Stromal cell clones derived from FL are large in size (mean forward light scatter intensity [mFSC] of 450), express the surface antigen Thy-1 but not Sca-1 and 6 out of 6 are able to differentiate into fat accumulating adipocytes. BM derived stromal cell clones are either small (mFSC of 250) or large (mFSC of 450), express Sca-1 but not Thy-1 and only 2 out of 7 differentiate towards adipocytes. Heterogeneity in terms of vascular adhesion molecule-1, intracellular adhesion molecule-1 and heat stable antigen expression was found among the different cell clones. Functional assays using long- and short-term cocultures of stromal and hematopoietic calls revealed: (1) the capacity of 8 out of 12 stromal cell clones to support the expansion of primitive hematopoietic progenitors (colony forming unit spleen day 12) more than 10 weeks. Fat accumulation but not expression of stem cell factor by stromal cells did correlate with this supportive function. (2) Better support of granulocyte maturation and proliferation by BM- compared to FL-derived stromal cell clones. However, stromal cell clones from both organs expressed macrophage-colony stimulating factor. (3) The ability of 4 out of 12 stromal cell clones (derived from both, FL and BM) to support the expansion of Interleukin-7 dependent pre-B cells from the BM. Pre-B cell growth stimulating factor was not restricted to supporters. (4) Mutual exclusiveness of myeloid and lymphoid support in that a given stromal cell clone supported either pre B-cell or granulocyte expansion. Experiments comparing the support of BM- and FL-derived hematopoietic progenitors showed identical responses of late (B220+/c-kit-) but strikingly different responses of early (B220+/c-kit+) pre-B cells, revealing different proliferation requirements for FL- versus BM- derived early pre-B cells in vitro.