There are a number of instances in which the use of live cell imaging has provided critical insight into cellular and tissue function. It has therefore become a requisite analytical tool for use in cell biology, neurobiology, and developmental biology, as well as a routine method practiced in many biomedical research laboratories. The technical requirements for performing live cell imaging include a capacity to engineer appropriate cells for the imaging experiment and access to a digital image acquisition system. The availability of fluorescent protein (FP) technology allows the molecular specificity of fluorescent markers to be used in a genetically encoded manner. In this article, we describe techniques for the generation and maintenance of fluorescently labeled cells and their use in live cell imaging experiments.