Enhancer of zeste homolog 2 (EZH2), a catalytic component of polycomb repressive complex 2 (PRC2), epigenetically regulates chromatin structure and gene expressions through tri-methylation at histone H3K27 and recruitment of DNA methyltransferases for gene silencing. Despite extensive studies of the role of EZH2 in cancer progression and malignancy, increasing evidence also suggest that EZH2 plays a critical role in stem cells renewal, maintenance, and differentiation into specific cell lineages. Here, we review the updated information regarding how EZH2 contributes to stem cell maintenance, cell lineage determination, including myogenesis, adipogenesis, osteogenesis, neurogenesis, hematopoiesis, lymphopoiesis, epidermal differentiation and hepatogenesis, and how EZH2 is regulated by phosphorylation and microRNAs in these processes.