The nucleolus is a non-membrane-bound nuclear organelle found in all eukaryotes. It is the quintessential 'RNA-seeded' nuclear body, forming around specific chromosomal features called nucleolar organizing regions that contain arrays of ribosomal DNA. Assembly is triggered by activation of RNA polymerase I-mediated transcription and regulated in mammalian cells in a cell cycle-dependent manner. Although the nucleolus is best known for its role in coordinating ribosome biogenesis, biochemical and proteomic analyses have revealed a much wider functional complexity than previously appreciated, including roles in cell cycle regulation, DNA damage sensing and repair, pre-mRNA processing, telomere metabolism, processing of non-coding RNAs, and coordination of the cellular response to various stresses. Despite these advances, much remains to be learned about the full range of biological processes that occur within, or involve, this organelle and how its assembly/disassembly and functional reorganization in response to various stimuli are regulated. Here, we review the impact of recent studies that provide major insights into these fundamental questions, and we highlight the therapeutic potential of targeting nucleolar pathways.