The release of neurotransmitters from presynaptic terminals is a strongly Ca2+-dependent process controlled by synaptotagmins, especially by their C2B domains. Biochemical measurements have reported Ca2+ affinities of synaptotagmin too low to account for synaptic function. However, binding of the C2B domain to the membrane phospholipid PI(4,5)P2 increases the Ca2+ affinity and vice versa, indicating a positive allosteric stabilization of simultaneous binding. Here, we construct a mathematical model of the release-triggering mechanism of synaptotagmin based on measured Ca2+/PI(4,5)P2 affinities and reported protein copy numbers. The model reproduced the kinetics of synaptic transmission observed at the calyx of Held over the full range of Ca2+ stimuli, with each C2B domain crosslinking Ca2+ and PI(4,5)P2 lowering the energy barrier for fusion by 4.85 kBT. The allosteric stabilization of simultaneous Ca2+ and PI(4,5)P2 binding was crucial to form multiple crosslinks which enabled fast fusion rates. Only three crosslinking C2B domains were needed to reproduce physiological responses, but high copy numbers per vesicle sped up the collision-limited formation of crosslinks. In silico evaluation of theoretical mutants revealed that affection of the allosteric properties might be a determinant of the severity of synaptotagmin mutations and may underlie dominant-negative, disease-causing effects. We conclude that allostericity is a crucial feature of synaptotagmin action.