INTRODUCTION: Mesenchymal stem cells (MSC) are an attractive cell source for cartilage and bone tissue engineering given their ability to differentiate into chondrocytes and osteoblasts. However, the common origin of these two specialized cell types raised the question about the identification of regulatory pathways determining the differentiation fate of MSC into chondrocyte or osteoblast. METHODS: Chondrogenesis, osteoblastogenesis and adipogenesis of human and mouse MSC were induced using specific inductive culture conditions. Expression of promyelocytic leukemia zinc-finger (PLZF) or differentiation markers in MSC was determined by RT-qPCR. PLZF-expressing MSC were implanted in a mouse osteochondral defect model and the neotissue was analyzed by routine histology and micro-computed tomography. RESULTS: We found out that PLZF is not expressed in MSC and its expression at early stages of MSC differentiation is the mark of their commitment toward the three main lineages. PLZF acts as an upstream regulator of both Sox9 and Runx2 and its overexpression in MSC enhances chondrogenesis and osteogenesis while it inhibits adipogenesis. In vivo, implantation of PLZF-expressing MSC in mice with full thickness osteochondral defects resulted in the formation of a reparative tissue resembling cartilage and bone. CONCLUSIONS: Our findings demonstrate that absence of PLZF is required for stemness maintenance and its expression is an early event at the onset of MSC commitment during the differentiation processes of the three main lineages.