The thymus provides a microenvironment that induces the differentiation of T-progenitor cells into functional T cells and that establishes a diverse yet self-tolerant T-cell repertoire. However, the mechanisms that lead to the development of the thymus are incompletely understood. We report herein the results of screening for genes that are expressed in the third pharyngeal pouch, which contains thymic primordium. Polymerase chain reaction (PCR)-based cDNA subtraction screening for genes expressed in microdissected tissues of the third pharyngeal pouch rather than the second pharyngeal arch yielded one transcription factor, MafB, which was predominantly expressed in CD45(-)IA(-)PDGFRalpha(+) mesenchymal cells and was detectable even in the third pharyngeal pouch of FoxN1-deficient nude mice. Interestingly, the number of CD45(+) cells that initially accumulated in the embryonic thymus was significantly decreased in MafB-deficient mice. Alterations of gene expression in the embryonic thymi of MafB-deficient mice included the reduced expression of Wnt3 and BMP4 in mesenchymal cells and of CCL21 and CCL25 in epithelial cells. These results suggest that MafB expressed in third pharyngeal pouch mesenchymal cells critically regulates lymphocyte accumulation in the embryonic thymus.