Establishing synaptic connections between neurons is essential for neuronal circuitry and efficient brain communication While the role of neuronal activity in refining and maintaining synapses is well known, we focus on its role, specifically spontaneous neurotransmitter release, for synapse formation between dentate gyrus axons, the mossy fibres (MF), onto CA3 pyramidal cells in the hippocampus. Neurotransmitter release occurs through two primary modes: action potential induced release and spontaneous fusion of synaptic vesicles. These modes are underpinned by distinct pools of vesicles, exhibiting varying cycling patterns during neuronal development. Predominantly spontaneous cycling in young neurons, transitions to the mature phenotype characterised by robust evoked release. Notably, existing studies suggests that evoked release is not important for synapse formation. Conversely, our previous work in dissociated hippocampal neurons indicated a potential role for spontaneous release in dendritic arborisation. However, whether it is important for synapse formation, particularly in vivo, remains unknown. This study utilises inducible conditional mouse models to manipulate spontaneous release levels in granule cells during the early developmental window characterised by high spontaneous cycling. Loss of synaptotagmin 1, the sensor for fast, evoked release, leads to loss of evoked release and enhanced spontaneous release. Conversely, loss of Munc18-1, a key component of the synaptic vesicle fusion complex, abolishes all release. Selective deletion of these proteins, at different developmental stages in vivo, revealed distinct effects on size and density of synaptic connections, indicating a crucial role for spontaneous release in shaping the formation of this circuit.