Enhancers are the primary regulatory DNA sequences in eukaryotes and are mostly located in the non-coding sequences of genes, namely, intergenic regions and introns. The essential characteristic of an enhancer is the ability to activate proximal genes, e.g., a reporter gene in a reporter assay, regardless of orientation, relative position, and distance from the gene. These characteristics are ascribed to the interaction (spatial proximity) of the enhancer sequence and the gene promoter via DNA looping, discussed in the latter part of this chapter.Developmentally regulated genes are associated with multiple enhancers carrying distinct cell and developmental stage specificities, which form arrays on the genome. We discuss the array of enhancers regulating the Sox2 gene as a paradigm. Sox2 enhancers are the best studied enhancers of a single gene in developmental regulation. In addition, the Sox2 gene is located in a genomic region with a very sparse gene distribution (no other protein-coding genes in ~1.6 Mb in the mouse genome), termed a "gene desert," which means that most identified enhancers in the region are associated with Sox2 regulation. Furthermore, the importance of the Sox2 gene in stem cell regulation and neural development justifies focusing on Sox2-associated enhancers.