The basic helix-loop-helix (bHLH) transcription factor Math1 and its orthologs are fundamental for proper development of various neuronal subpopulations, such as cerebellar granule cells, D1 interneurons in the spinal cord, and inner ear hair cells. Although crucial for neurogenesis, the mechanisms by which Math1 specifically recognizes its direct targets are not fully understood. To search for direct and indirect target genes and signaling pathways controlled by Math1, we analyzed the effect of Math1 knockout on the expression profile of multiple genes in the embryonic cerebellum. Eighteen differentially expressed transcripts were identified and found to belong to a few developmentally-related functional groups, such as transcriptional regulation, proliferation, organogenesis, signal transduction, and apoptosis. Importantly, genomic analysis of E-box motifs has identified a significant enrichment and clustering of MATH1-binding E-boxes only in a subset of differentially expressed genes (Nr2f6, Hras1, and Hes5) in both mouse and man. Moreover, Math1 was shown by chromatin immunoprecipitation (ChIP) to bind, and by a luciferase reporter assay to activate transcription, of an upstream genomic fragment of Nr2f6. Taken together, we propose that when putative direct targets of Math1 are being selected for detailed studies on DNA microarray hybridization, the enrichment and clustering of binding E-boxes in multiple species may be helpful criteria. Our findings may be useful to the study of other bHLH transcription factors, many of which control the development of the nervous system.