Allogeneic bone grafts are a promising material for bone implantation due to reduced operative trauma, reduced blood loss, and no donor-site morbidity. Although human decellularized allogeneic bone (hDCB) can be used to fill bone defects, the research of revitalizing hDCB blocks with human mesenchymal stem cells (hMSCs) for osteochondral regeneration is missing. The hMSCs derived from bone marrow, adipose tissue, and Wharton’s jelly (BMMSCs, ADMSCs, and UMSCs, respectively) are potential candidates for bone regeneration. This study characterized the potential of hDCB as a scaffold for osteogenesis and chondrogenesis of BMMSCs, ADMSCs, and UMSCs. The pore sizes and mechanical strength of hDCB were characterized. Cell survival and adhesion of hMSCs were investigated using MTT assay and F-actin staining. Alizarin Red S and Safranin O staining were conducted to demonstrate calcium deposition and proteoglycan production of hMSCs after osteogenic and chondrogenic differentiation, respectively. A RT-qPCR was performed to analyze the expression levels of osteogenic and chondrogenic markers in hMSCs. Results indicated that BMMSCs and ADMSCs exhibited higher osteogenic potential than UMSCs. Furthermore, ADMSCs and UMSCs had higher chondrogenic potential than BMMSCs. This study demonstrated that chondrogenic ADMSCs-or UMSCs-seeded hDCB might be potential osteochondral constructs for osteochondral regeneration.