Extracellular environment is a physical support that is critical to cell adhesion, migration, and differentiation. In this work, cell-derived matrices (CDMs) were obtained by separately culturing fibroblasts, preosteoblasts, and chondrocytes. The cells were grown on a coverslip and subjected to decellularization using detergents and enzymes. The resulting matrices were named fibroblast-derived matrix (FDM), preosteoblast-derived matrix (PDM), and chondrocyte-derived matrix (CHDM). We hypothesize that the unique compositional and structural feature of each CDM provides cells with a distinct microenvironment capable of functioning as a different signaling cue in the regulation of preosteoblast and rat bone marrow mesenchymal stromal cell (BMSC) osteogenic differentiation. SEM images show that each cell type creates its unique surface texture in a fibrillar structure. Three major macromolecules, fibronectin, type I collagen, and laminin, were clearly identified using both immunofluorescence and Western blot, in which FDM exhibited a much stronger signal of each ECM component than that of PDM or CHDM. For early cell morphology, BMSCs on the CDMs were highly elongated in a spindle-like shape. Both preosteoblasts and BMSCs proliferated well on CDMs comparable to the control. Once preosteoblasts were cultured for 2 weeks, their osteogenic activity was significantly different depending on the type of CDM. Using Alizarin red and von Kossa staining, we found that the cells on the FDM were much more osteogenic than the other groups. Furthermore, FDM was the most effective in upregulating the osteogenic markers, such as alkaline phosphatase (ALP), osteopontin, osteocalcin, and type I collagen. In particular, we observed a 2.5-fold increase in ALP activity with FDM compared to that of control and CHDM. In stark contrast, CHDM was very poor in stimulating osteogenic differentiation of preosteoblasts. Interestingly, these results were reproducible with the use of BMSCs, which are much more heterogeneous in cell populations than preosteoblasts. CHDM was still very weak in triggering the osteogenesis of BMSCs, whereas both FDM and PDM were equally competitive. This study demonstrates that a combination of factors (surface texture and composition) shape a unique cellular microenvironment, which serves as a physical cue toward the osteogenic differentiation of preosteoblasts and BMSCs.