Co-ordinated progression through the cell cycle is essential for the maintenance of genomic integrity. Several checkpoint mechanisms guarantee that the next step in cell cycle progression is only entered after error-free completion of the previous phase. Cell cycle deregulation caused by changes in 14-3-3 expression has been implicated in cancer formation. 14-3-3 proteins function at several key points in G₁/S- and G₂/M-transition by binding to regulatory proteins and modulating their function. In most cases, the association with 14-3-3 proteins requires a specific phosphorylation of the protein ligand and mediates cell cycle arrest. 14-3-3 binding may lead to cytoplasmic sequestration of the protein ligand but may also have other functional consequences. The 14-3-3σ gene is induced by p53 and its product inhibits G₂/M progression by cytoplasmatic sequestration of CDC2-cyclin B complexes. In addition, 14-3-3 proteins have been implicated in the transcriptional regulation of CDK-inhibitors as they modulate the transcription factors p53, FOXO and MIZ1. Effects of 14-3-3 proteins on cell cycle progression and the regulation of 14-3-3 activity during the cell cycle are reviewed in this chapter.