During embryogenesis, transcription factor GATA2 is expressed in a variety of distinct cell types, and earlier experiments showed that GATA2 is a vital regulator of both hematopoiesis and urogenital development. Despite the fact that GATA2 is expressed early and abundantly in the nervous system, there has been no demonstration of its direct participation in neurogenesis. We show here that GATA2 is expressed in the ventral spinal cord exclusively in newly generated V2 interneurons, suggesting that GATA2 might be required for the generation of this discrete neuronal population. Proof for this hypothesis was provided by showing that the number of cells expressing V2 neuronal markers was drastically diminished in gata2 null mutant embryos. The tissue-specific enhancer that directs gata2 transcription specifically in V2 neurons was localized to a 190 bp intragenic element lying within gata2 intron 5, and this element is both necessary and sufficient to confer GATA2 spinal cord expression. The identification of a V2-specific enhancer should allow fundamental new insight into the genetic hierarchy of regulatory events that govern neurogenesis in a well-defined cell lineage.