Bone marrow- and adipose tissue-derived stromal cells (BMSCs and ASCs, respectively) exhibit a similar capacity for osteogenic differentiation in vitro, but it is unclear whether they share a common differentiation process, because they originate from different tissues. The aim of this study was to explore BMSC and ASC osteogenic differentiation by focusing on the expression of extracellular matrix-related genes (ECMGs), which play a crucial role in osteogenesis and bone tissue regeneration in vivo. We characterized the gene expression profiles of BMSCs and ASCs using a custom complementary deoxyribonucleic acid microarray containing 55 ECMGs. Undifferentiated BMSCs and ASCs actively expressed a wide range of ECMGs. Once BMSCs and ASCs were placed in an osteogenic differentiation medium, 24 and 17 ECMGs, respectively, underwent considerable downregulation over the course of the culture period. The remaining genes were maintained at a similar expression level to corresponding uninduced cell cultures. Although the suppression phenomenon was consistent irrespective of stromal cell origin, collagen (COL)2A1, COL6A1, COL9A1, parathyroid hormone receptor, integrin (INT)-beta3, and TenascinX genes were only downregulated in osteogenic BMSCs, whereas COL1A2, COL3A1, COL4A1, COL5A2, COL15A1, osteopontin, osteonectin, and INT-beta1 genes were only downregulated in osteogenic ASCs. During this time period, cell viability was sustained, suggesting that the observed downregulation did not occur by selection and elimination of unfit cells from the whole cell population. These data suggest that osteogenically differentiating BMSCs and ASCs transition away from a diverse gene expression pattern, reflecting their multipotency toward a configuration specifically meeting the requirements of the target lineage. This change may serve to normalize gene expression in mixed populations of stem cells derived from different tissues.