Human bone marrow derived mesenchymal stem/stromal cells (MSC) are multipotential and have the ability to differentiate into osteoblasts, myoblasts, adipocytes and chondrocytes in vitro and in vivo4 3 2 1. MSC differentiation is regulated by different signalling molecules and transcription factors that regulate the Wnt, BMP and TGFβ pathways, which help determine lineage commitment. Recently, epigenetic modifiers which modify chromatin have been shown to be important for allowing the activation or repression of transcription factors and pathways which regulate MSC lineage commitment 6 5. One epigenetic modifier methyltransferase, Enhancer of Zeste homology 2 (EZH2), tri-methylates histone three lysine twenty seven (H3K27me3) on chromatin, leading to the compaction of chromatin and repression of genes. Epigenetic modifier lysine demethylase 6A (KDM6A) can remove this EZH2 mediated H3K27me3 modification, allowing the recruitment of factors involved in the unravelling of chromatin and activation of genes. In this study we demonstrate an inverse relationship between Ezh2 and Kdm6a transcript levels during differentiation of human bone marrow derived MSC. Retroviral mediated enforced expression of Ezh2 in MSC promoted adipogenic and inhibited osteogenic differentiation in vitro, whereas Kdm6a inhibited adipogenesis and promoted osteogenic differentiation. Pharmacological inhibition of EZH2 activity or siRNA mediated knockdown of Ezh2 gene expression in human MSC resulted in decreased adipogenesis and increased osteogenesis. Conversely, siRNA knockdown of Kdm6a gene expression in MSC led to increased adipogenesis and decreased osteogenesis. Both EZH2 and KDM6A were shown to affect expression of master regulatory genes involved in adipogenesis and osteogenesis and H3K27me3 on the promoters of master regulatory genes. These findings demonstrate an important epigenetic switch centred on H3K27me3 which dictates MSC lineage determination