Cellular metabolism has recently emerged as a key regulator of stem cell behavior. Various studies have suggested that metabolic regulatory mechanisms are conserved in different stem cell niches, suggesting a common level of stem cell regulation across tissues. Although the balance between glycolysis and oxidative phosphorylation has been shown to be distinct in stem cells and their differentiated progeny, much less is known about lipid metabolism in stem cell regulation. In this Review, we focus on how stem cells are affected by two major lipid metabolic pathways: the build-up of lipids, called de novo lipogenesis, and the breakdown of lipids, called fatty acid beta-oxidation. We cover the recent literature on hematopoietic stem cells, intestinal stem cells, neural stem/progenitor cells and cancer stem cells, where these two lipid pathways have been studied in more depth.