We cultured MSCs on an ECM made by bone marrow cells to attempt to reconstitute the MSC niche. This ECM promoted replication of mesenchymal progenitors and retention of their multipotentiality. We conclude that the marrow ECM facilitates expansion of mesenchymal progenitors and hypothesize that it plays an important role in the maintenance of MSC stemness. Mesenchymal colony-forming cells of the bone marrow comprise mesenchymal stem cells (MSCs) and their transit amplifying progeny, which we term mesenchymal colony-forming units (MCFUs). These progenitors undergo self-renewal and can differentiate into many different cell types including osteoblasts. However, they lose their unique properties when cultured on tissue culture plastic. This indicates that a critical feature of the marrow microenvironment that facilitates retention of stem cell properties is missing in such culture systems. In other tissues, the extracellular matrix (ECM) forms part of the specialized niche that controls stem cell behavior. Therefore, we examined whether a marrow cell-derived ECM promotes retention of the stem cell characteristics of MCFUs in vitro. A cell-free ECM was prepared from cultured murine marrow adherent cells. The replication and multipotentiality of murine MCFUs maintained on this marrow cell-derived ECM were examined in vitro and in vivo and compared with the behavior of MCFUs maintained on plastic. The marrow cell-derived ECM was made up of collagen types I, III, and V, syndecan-1, perlecan, fibronectin, laminin, biglycan, and decorin, similar to the composition of the marrow ECM. This ECM preparation promoted MCFU replication, restrained their "spontaneous" differentiation toward the osteoblast lineage, and preserved their ability to differentiate into osteoblasts or adipocytes. Moreover, transplantation of MCFUs expanded on the marrow cell-derived ECM into immunocompromised mice generated five times more bone and eight times more hematopoietic marrow compared with MCFUs expanded on plastic. The marrow ECM facilitates expansion of MCFUs in vitro while preserving their stem cell properties. We hypothesize that the ECM made by bone marrow cells plays an important role in the maintenance of MSC function.