Two mitotic Cyclins, A and B, exist in higher eukaryotes, but their specialised functions in mitosis are poorly understood. Using degron tags we analyse how acute depletion of these proteins affects mitosis. Loss of Cyclin A in G2-phase prevents the initial activation of Cdk1. Cells lacking Cyclin B can enter mitosis and phosphorylate most mitotic proteins, because of parallel PP2A:B55 phosphatase inactivation by Greatwall kinase. The final barrier to mitotic establishment corresponds to nuclear envelope breakdown that requires a decisive shift in the balance of Cdk1 and PP2A:B55 activity. Beyond this point Cyclin B/Cdk1 is essential to phosphorylate a distinct subset mitotic Cdk1 substrates that are essential to complete cell division. Our results identify how Cyclin A, B and Greatwall coordinate mitotic progression by increasing levels of Cdk1-dependent substrate phosphorylation. 3 Main TextCdk1 phosphorylates over 1000 proteins 1,2 within the brief 20-30 minute window of mitotic entry, triggering centrosome separation and chromosome condensation in prophase, followed by nuclear envelope breakdown (NEBD) and mitotic spindle formation in prometaphase, and the alignment of bi-oriented sister chromatids at the metaphase plate 3 . Binding of a Cyclin partner is critical for allosteric activation of CDKs. Two families of mitotic Cyclins, termed A and B, work with Cdk1 to orchestrate mitotic entry in higher eukaryotes 4,5 . Despite the central importance of these proteins for cell cycle control, the functional specialisation of mammalian A and B-type Cyclins remains unclear. Following the depletion of maternal pools of early embryonic Cyclin A1 and B3, somatic mammalian cells express one A-type Cyclin, A2, and two B-type cyclins, B1 and B2. Genetic depletion in mice suggests an essential role for Cyclin A2 for development, but not for embryonic fibroblast proliferation 6 . Depletion of Human Cyclin A2 by siRNA delays mitotic entry, and this is further enhanced by co-depletion of Cyclin B1 7-9 . Likewise, work in mammalian cell extracts documented how Cyclin A synergises with Cyclin B to control the mitotic entry threshold at the level for Cdk1 activation 10 . A mechanism involving Plk1 activation has been suggested 11,12 , although an essential role of Plk1 in the G2/M transition remains contentious 13 . Likewise, work in mammalian cell extracts documented how Cyclin A synergises with Cyclin B to control the mitotic entry threshold at the level for Cdk1 activation 10 . A confounding factor in the genetic analysis of Cyclin A2 has been its dual role in S-phase and mitosis 14 , making it difficult to directly investigate G2 specific defects. Murine Cyclin B1 is essential for development 15 and critical for mitotic entry in early mouse embryos 16 . Conversely, mice lacking Cyclin B2 live healthily, without apparent defects 15 . These results stand in stark contrast to observations from experiments involving siRNA depletion of B-type Cyclins in human cancer cell lines that show surprisingly mild mitotic entry defects 9,1...