The vertebrate spinal cord has long served as a useful system for studying the pattern of cell differentiation along the dorsoventral (d/v) axis. In this paper, we have defined the expression of several classes of genes expressed in restricted d/v domains in the intermediate region (IR) of the mouse spinal cord, in which most interneurons are generated. From this analysis, we have found that spinal cord interneurons and their precursors express unique combinations of transcription factors and Notch ligands at the onset of their differentiation. The domains of expression of a number of different classes of genes share similar boundaries, indicating that there could be a basic subdivision of the ventral IR into four distinct regions. This differential gene expression suggests that spinal cord interneurons acquire unique identities early in their development and that Notch signaling mechanisms may participate in the determination of cell fate along the d/v axis. Gene expression studies inEngrailed-1(En-1) mutants showed thatEn-1-expressing and other closely positioned classes of neurons do not require the homeodomain protein En-1 for their early pattern of differentiation. Rather, it is suggested thatEn-1may function to distinguish a subset of interneurons during the later maturation of the spinal cord.