Although S100 calcium binding (S100) proteins are unique calcium-binding proteins with time-dependent patterns of expression, their roles in vertebrate development and organogenesis remain unknown. Here we identify that a member of s100a subfamily, is necessary for epithelial development of the pharyngeal arches and the gut tube. We generated transgenic zebrafish in which the expression of enhanced green fluorescent protein (EGFP) is under the control of a 4 kb upstream regulatory element of s100 gene. The EGFP positive cells widely distribute along the surface of the transgenic embryos during early development and then are restricted to the epithelial layer covering pharyngeal arches, olfactory placodes and the gut tube. The EGFP positive cells in the intestinal epithulium display polarized enteroendocrine cell morphology and produce pancreatic peptide. We show that knockdown using morpholinos (MOs) of the S100a gene causes curved embryo bodies, reduction of fgf3 expression and loss of posterior pharyngeal arches. Intestinal endocrine cells expressing nxk2.2a, a homeodomain transcriptional factor marking enteroendocrine cells, are absent in the s100a gene deficient embryos. Moreover, knockdown of nkx2.2a abolishes a subset of the s100a subfamily-expressing entroendocrine cells, whereas reducing the s100a subfamily in nkx2.2a-deficient embryos completely eliminates these enteroendocrine cells, revealing a regulatory complexity during enteroendocrine cell formation. We also established the germline mutant of the s100a gene using transcription activator-like effector nuclease (TALEN). All these findings thus establish the first known roles for S100 proteins during vertebrate development, and provide biological insights into the roles of epithelial patterning in development of the pharyngeal arches and the intestine.