The stromal cell layer is believed to play an important role in long-term human bone marrow cultures (LTHBMCs). At present, neither the role that the stromal cell extra-cellular matrix (ECM) plays in influencing stroma behavior is well understood nor are the effects of stroma aging. Rapid medium exchanged LTHBMCs were established on surfaces precoated with human natural fibronectin and type 1 rat tail collagen. Although initial adhesion of hematopoietic cells was improved by the presence of both ECMs, the overall progenitor and nonadherent cell productivity was not improved nor did the stroma grow to confluency faster. Thus, the ECMs used did not significantly influence the cell productivity of LTHBMCs. To examine the influence of stromal cell layer aging, conditioned medium was obtained from the first two weeks of LTHBMCs that was subsequently concentrated and used as a medium supplement in a second set of slowly exchanged LTHBMCs. The presence of the concentrated conditioned medium (conCM) enhanced the production of nonadherent cells three-fold compared with control over an eight week culture period. Control cultures that were exposed to conCM after 4 weeks in culture significantly improved their cell productivity during the latter 4 weeks of culture compared with control. The productivity of cultures exposed to conCM for 4 weeks dropped significantly when unsupplemented medium was used for the latter 4 weeks of culture. Interestingly, phytohemagglutin-stimulated leukocyte-conditioned medium stimulated LTHMBCs in a similar fashion, as did conditioned medium from early LTHBMCs. Taken together, these results strongly suggest that the stromal cell layer does produce important factors for active hematopoiesis during its growth to confluence.