Human gingival stem cells (HGSC) can be easily isolated and manipulated in culture to investigate their multipotency. Osteogenic differentiation of bone marrow derived mesenchymal stem cells (BMMSC) has been well documented. HGSC derive from neural crests, however, and their differentiation capacity has not been fully established. The aim of the present report was to investigate whether HGSC can be induced to differentiate to osteoblasts and chondrocytes. HGSC were cultured either in a classical monolayer culture or in 3-dimensional floating micromass pellet cultures in specific differentiation media. HGSC differentiation to osteogenic and chondrogenic lineages was determined by protein and gene expression analyses, and also by specific staining of cells and tissue pellets. HGSC cultured in osteogenic differentiation medium showed induction of Runx2, alkaline phosphatase and osterix expression, and subsequently formed mineralized nodules consistent with osteogenic differentiation. Interestingly, HGSC micromass cultures maintained in chondrogenic differentiation medium showed SOX9-dependent differentiation to both chondrocyte and synoviocyte lineages. Chondrocytes at different stages of differentiation were identified by gene expression profiles and by histochemical and immunohistochemical staining. In 3-week-old cultures, peripheral cells in the micromass cultures organized in layers of cuboidal cells with villous structures facing the medium. These cells were strongly positive for cadherin-11, a marker of synoviocytes. In summary, the findings indicate that HGSC have the capacity to differentiate to osteogenic, chondrogenic and synoviocyte lineages. Therefore, HGSC could serve as an alternative source for stem cell therapies in regenerative medicine for patients with cartilage and joint destructions, such as observed in rheumatoid arthritis.