ABSTRACT The nucleolus is a membrane-less subnuclear compartment known for its role in ribosome biogenesis. However, emerging evidence suggests that nucleolar function extends beyond ribosome production and is particularly important during mammalian development. Nucleoli are dynamically reprogrammed post-fertilisation: totipotent early mouse embryos display non-canonical, immature nucleolar precursor bodies, and their remodelling to mature nucleoli is essential for the totipotency-to-pluripotency transition. Mounting evidence also links nucleolar disruption to various pathologies, including embryonic lethality in mouse mutants for nucleolar factors, human developmental disorders and observations of nucleolar changes in disease states. As well as its role in ribogenesis, new findings point to the nucleolus as an essential regulator of genome organisation and heterochromatin formation. This Review summarises the varied roles of nucleoli in development, primarily in mammals, highlighting the importance of nucleolar chromatin for genome regulation, and introduces new techniques for exploring nucleolar function.