A major challenge in systems neuroscience is to perform precise molecular genetic analyses of a single neuronal population in the context of the complex mammalian brain. Existing technologies for profiling cell type–specific gene expression are largely limited to immature or morphologically identifiable neurons. In this study, we developed a simple method using fluorescent activated cell sorting (FACS) to purify genetically labeled neurons from juvenile and adult mouse brains for gene expression profiling. We identify and verify a new set of differentially expressed genes in the striatonigral and striatopallidal neurons, two functionally and clinically important projection neuron subtypes in the basal ganglia. We further demonstrate that Ebf1 is a lineage-specific transcription factor essential to the differentiation of striatonigral neurons. Our study provides a general approach for profiling cell type–specific gene expression in the mature mammalian brain and identifies a set of genes critical to the function and dysfunction of the striatal projection neuron circuit.