Objective: The present study was aimed to investigate the osteogenic potency of scaffold-free threedimensional (3D) spheres of periodontal ligament stem cells (PDLSCs). Study Design: The osteogenic potency of PDLSC spheres was determined by the ability to form mineralization and to express key osteogenesis-associated genes. The alkaline phosphatase (ALP) activity and the protein content of PDLSC spheres were also measured. Results: The 3D sphere developed its osteogenic potency in a timedependent manner, containing approximately 10 folds higher of the mineralization, 5 folds higher protein content and 4 folds greater ALP activity than those in the controls. The expression of key osteogenic genes was also up-regulated in the 3D PDLSC spheres. Cellular outgrowth was observed when reintroduced into 2D culture. Conclusion: PDLSCs were able to undergo osteogenic differentiation in a scaffold-free 3D culture, producing bone-like mineralization in vitro. This suggests, at least in vitro, the osteogenic potency of the 3D PDLSC spheres.