Information processing in the nervous system relies on properly localized and organized synaptic structures at the correct locations. The formation of synapses is a long and intricate process involving multiple interrelated steps. Decades of research have identified a large number of molecular components of the presynaptic compartment. In addition to neurotransmitter-containing synaptic vesicles, presynaptic terminals are defined by cytoskeletal and membrane specializations that allow highly regulated exo- and endocytosis of synaptic vesicles and that maintain precise registration with postsynaptic targets. Functional studies at multiple levels have revealed complex interactions between the transport of vesicular intermediates, the presynaptic cytoskeleton, growth cone navigation, and synaptic targets. With the advent of finer anatomical, physiological, and molecular tools, great insights have been gained toward the mechanistic dissection of functionally redundant processes controlling the specificity and dynamics of synapses. This review highlights the recent findings pertaining to the cellular and molecular regulation of presynaptic differentiation.