During development of the central nervous system (CNS), cycling uncommitted progenitor cells give rise to a variety of distinct neuronal and glial cell types. As these different cell types are born they progress from newly specified cells to fully differentiated neurons and glia. In order to define the developmental processes of individual cell types, single cell expression profiling was carried out on developing ganglion and amacrine cells of the murine retina. Individual cells from multiple developmental stages were isolated and profiled on Affymetrix oligonucleotide arrays. Two-color fluorescent in situ hybridization on dissociated retinas was used to verify and extend the microarray results by allowing quantitative measurements of a large number of cells coexpressing two genes. Together, these experiments have yielded an expanded view of the processes underway in developing retinal ganglion and amacrine cells, as well as several hundred new marker genes for these cell types. In addition, this study has allowed for the definition of some of the molecular heterogeneity both between developing ganglion and amacrine cells and among subclasses of each cell type.