Neuronal function depends on the accurate wiring between pre- and postsynaptic cells. Determining the mechanisms underlying precision in neuronal connectivity is challenging because of the complexity of the nervous system. In diverse parts of the nervous system, regions of synaptic contact are organized into distinct parallel layers, or laminae, that are correlated with distinct functions. Such an arrangement enables the development of synapse specificity to be more readily investigated. Here, we present an overview of the developmental mechanisms that are thought to underlie the formation of synaptic layers in the vertebrate retina, a highly laminated CNS structure. We will contrast the roles of activity-dependent and activity-independent mechanisms in establishing functionally discrete sublaminae in the inner retina, where circuits involving many subtypes of retinal neurons are assembled precisely. In addition, we will discuss new optical imaging approaches for elucidating how retinal synaptic lamination occurs in vivo.