Sixfamily genes encode homeobox transcription factors, and a deficiency in them leads to abnormal structures of the sensory organs. In a previous paper, Six1 was reported to be expressed in the taste bud-bearing lingual papillae of mice, and loss of Six1 affected the development of these gustatory papillae. We show here that embryos lacking both Six1 and Six4 revealed more severe abnormalities than those lacking Six1 alone during morphogenesis of their gustatory papillae. By in situ hybridization, Six4 was shown to be broadly distributed in the epithelium of the lateral lingual swellings at embryonic day (E) 11.5, and in the tongue epithelium, mesenchyme, and muscles at E12.5. From E14, Six4 was similar in expression pattern to Six1, as previously reported. In the fungiform papillae, Six4 was expressed in the epithelium at E14–E16.5. In the circumvallate and foliate papillae, Six4 expression was observed in the trench wall of these papillae at E15.5-P0. Although Six4-deficient mice had no abnormalities, Six1/Six4-deficient mice showed distinct morphological changes: fusion of the lateral lingual swellings was delayed, and the tongue was poorly developed. The primordia of fungiform papillae appeared earlier than those in the wild-type or Six1-deficient mice, and the papillae rapidly increased in size; thus fusion of each papilla was evident. The circumvallate papillae showed severe defects; for example, invagination of the trenches started asymmetrically, which resulted in longer and shorter trenches. The foliate papillae elevated initially, and showed stunted trenches. Therefore, Six1 and Six4 function synergistically to form gustatory papillae during development of the tongue.