The complexity of the brain often requires neuroscientists to use a simpler system for experimental manipulations and observations. One powerful approach is to generate a primary culture by dissecting nervous system tissue, dissociating it into single cells, and growing those cells in vitro. Primary cultures make neurons and glia easily accessible to the experimental tools required for techniques like genetic manipulation and time-lapse imaging. Furthermore, these cultures represent a highly controllable environment in which to study complex phenomena such as cell-cell interactions. This video provides an overview of the major steps in producing primary neuronal cultures, which include selecting and dissecting the tissue of interest, mechanically and chemically breaking down the tissue to produce a single cell suspension, plating the cells, and maintaining the cultures in the appropriate media. Several example experiments are also presented to show how cultured cells can be used to investigate protein trafficking, morphological changes, and electrophysiology in living neurons.