Transient receptor potential channel M5 (Trpm5)-expressing cells, such as sweet, umami, and bitter taste cells in the oropharyngeal epithelium, solitary chemosensory cells in the nasal respiratory epithelium, and tuft cells in the small intestine, that express taste-related genes function as chemosensory cells. Previous studies demonstrated that Skn-1a/Pou2f3, a POU homeodomain transcription factor is expressed in these Trpm5-expressing chemosensory cells, and is necessary for their generation. Trpm5-expressing cells have recently been found in trachea, auditory tube, urethra, thymus, pancreatic duct, stomach, and large intestine. They are considered to be involved in protective responses to potential hazardous compounds as Skn-1a-dependent bitter taste cells, respiratory solitary chemosensory cells, and intestinal tuft cells are. In this study, we examined the expression and function of Skn-1a/Pou2f3 in Trpm5-expressing cells in trachea, auditory tube, urethra, thymus, pancreatic duct, stomach, and large intestine. Skn-1a/Pou2f3 is expressed in a majority of Trpm5-expressing cells in all tissues examined. In Skn-1a/Pou2f3-deficient mice, the expression of Trpm5 as well as marker genes for Trpm5-expressing cells were absent in all tested tissues. Immunohistochemical analyses demonstrated that two types of microvillous cells exist in trachea, urethra, and thymus, Trpm5-positive and Trpm5-negative cells. In Skn-1a/Pou2f3-deficient mice, a considerable proportion of Trpm5-negative and villin-positive microvillous cells remained present in these tissues. Thus, we propose that Skn-1a/Pou2f3 is the master regulator for the generation of the Trpm5-expressing microvillous cells in multiple tissues.