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Human periodontal ligament stem cells (HPLSCs) contribute to the regeneration of periodontal tissue because of their multilineage potential. Although monolayer cultures are commonly used in cell cultures, they inadequately overcome their low differentiation capacity. The use of spheroid cultures is expected to overcome the problem of mimicking the in vivo microenvironment. In this study, we assessed whether HPLSC spheroids are susceptible to osteogenic differentiation through the canonical Wnt/β-catenin signaling pathway. HPLSC spheroids were generated using low-binding plates. Osteogenic differentiation of monolayer-and spheroid-derived HPLSCs was induced by osteogenic induction medium. Increased expression levels of osterix and Runx2 and intense staining of alkaline phosphatase (ALP) activity were observed in spheroid-derived HPLSCs, as compared with monolayer-derived cells. During spheroid formation, the integrin pathway of cells composed of spheroids was activated through focal adhesion kinase (FAK), suggesting that this activation may induce susceptibility of the Wnt pathway in HPLSC spheroids. Wnt 3a stimulation increased the expression levels of β-catenin and T-cell factor (TCF), but decreased that of glycogen synthase kinase-3β. Wnt 3a-induced the expression of β-catenin and TCF was effectively decreased by Dickkopf-1 (Dkk-1), a Wnt antagonist. Wnt 3a stimulation also increased the expression levels of osterix and Runx2 which was accompanied by intense ALP staining, in HPLSC spheroids, whereas the addition of Dkk-1 decreased both expression levels and ALP staining. These findings indicate that HPLSC spheroids enhance osteogenic differentiation because cells, composed of spheroids, induce susceptibility of the canonical Wnt pathway, which is mediated by activation of the integrin/FAK pathway during spheroid formation.