The assembly of neural circuits in the developing brain is critical for the establishment of nervous system function. Investigation of this process is important to understand how connectivity changes across development, maturity and in diseases states. My thesis has applied techniques ranging from molecular biology to light and electron microscopy, specifically to study the assembly of the hippocampal mossy fiber synapse. The mossy fiber synapse exhibits unique structural and functional properties, making it an ideal system to study the differentiation of synapse specific properties. To investigate the structural maturation of the mossy fiber synapse at a level not previously possible, I have utilized the novel technique of serial blockface scanning electron microscopy. This technique allows the rapid acquisition of large volumes of perfectly aligned ultrastructural data for the three- dimensional reconstruction of local mossy fiber microcircuitry. Using volumes obtained from developing stratum lucidum neuropil, I have demonstrated 1) the range of pre and post-synaptic structural diversity at this synapse, 2) developmental refinement of structural features relevant to connectivity and 3) the relationship between pre and post-synaptic elements during the establishment of mature patterns of connectivity. The assembly of mossy fiber circuitry occurs entirely during postnatal development, a time during which both genetically determined and activity-dependent processes operate. To understand the molecular mechanisms underlying mossy fiber synapse maturation, I have investigated the role of the calcium-activated transcription factor NeuroD2 in this process. Using NeuroD2 null mice and in vivo single cell knockdown approaches, I find that 1) Development of mossy fiber synapse structure and function depends critically on NeuroD2 mediated transcription, 2) NeuroD2 acts post-synaptically to regulate the density of post-synaptic structure at the mossy fiber synapse in vivo, 3) NeuroD2 regulates the expression of PSD95 in the developing hippocampus and 4) Knockdown of PSD95 in vivo recapitulates the effect of NeuroD2 loss of function. These experiments indicate that NeuroD2 regulates a transcriptional program that is critical for the process of mossy fiber synapse maturation identified using serial blockface scanning electron microscopy