The self-renewal capacity of multipotent haematopoietic stem cells (HSCs) supports blood system homeostasis throughout life and underlies the curative capacity of clinical HSC transplantation therapies. However, despite extensive characterization of the HSC state in the adult bone marrow and embryonic fetal liver, the mechanism of HSC self-renewal has remained elusive. This Review presents our current understanding of HSC self-renewal in vivo and ex vivo, and discusses important advances in ex vivo HSC expansion that are providing new biological insights and offering new therapeutic opportunities.