Key determinants in the development of complex morphology and function are the cues present in a cell’s environment and its response to them. Primary among these extracellular factors is the presence and influence of neighboring cells. It is crucial, therefore, in studying development to be able to replicate in vitro these network-like conditions. This is especially true of neuroscience, tissue engineering, and clinical biology, where network formation and function are critical aspects of any investigation. Here we describe an easy and inexpensive technique based on microfluidics that provides a high degree of control in positioning and guiding cells, thereby enabling the laying down of desired cellular networks. This approach facilitates the study of synaptic connections where information is communicated between neurons. Such microscale devices are increasingly being employed for studying neurons in highly controlled environments wherein different regions of a network, or even a cell, are cultured in fluidically isolated compartments. Enhanced strategies such as highly regulated manipulations of fluid flow and physical guidance cues when combined with this compartmentalization provide an unparalleled degree of spatiotemporal control over the conformation of the neural network and the stimulation of synapses. This facilitates high-resolution investigations despite the cellular complexity. Consequently, the microfluidic culture platform presents an unparalleled context for unraveling the changes occurring at the microscale and nanoscale of synaptic connections, thereby aiding elucidation of the nuances of neuronal development, wiring, and function.