© 2018 Federation of European Biochemical Societies Cell surface glycans, which are tissue-specific and developmentally regulated, work as essential modulators in ligand–receptor interactions, binding to various signal ligands including Wnt, Hedgehog, fibroblast growth factors, epidermal growth factors, and bone morphogenetic proteins, as well as in cell–cell interactions and cell–extracellular matrix interactions. These signals are essential for the stemness and differentiation of various kinds of stem cells. In addition, the intracellular O-linked N-acetylglucosamine, a form of glycosylation found only on nuclear or cytoplasmic proteins, regulates core transcription factors of stemness and phosphorylation of downstream signal components. Therefore, various kinds of glycans regulate the stem cell status; the structures of many of which are evolutionarily conserved from Drosophila to mammals. Understanding the molecular mechanisms of glycans in stemness and differentiation is increasingly important for innovative clinical applications, as well as for basic research. This Review focuses on the roles of glycans in Drosophila tissue stem cells and mammalian pluripotent stem cells.