Osteoprotegerin (OPG), a conventional potent inhibitor of osteoclastogenesis, is being increasingly recognized for its diverse roles beyond bone metabolism. However, the cell-autonomous role of OPG in regulating the differentiation and fate of mesenchymal progenitor cells (MPCs) remains to be fully elucidated. To address this issue, OPG-knockout (OPG-KO) human induced pluripotent stem cell-derived MPCs were generated using CRISPR/Cas9 technology. Transcriptomics revealed that OPG deficiency fundamentally alters the functional landscape of the MPCs, with a significant downregulation of the pathways related to the extracellular matrix (ECM), cell adhesion, and structural signaling. Specifically, the expression of numerous key ECM components was broadly attenuated in OPG-KO MPCs. Such molecular disruption functionally translated into severely impaired osteogenic potential, characterized by a marked transcriptional attenuation of osteogenic markers and reduced matrix mineralization at the cell level. Collectively, our findings demonstrate that OPG is essential for maintaining the structural integrity of the MPC niche by regulating the expression of ECM-related genes, thereby promoting osteoblast differentiation.