Epithelial–mesenchymal interactions have essential roles in thymus organogenesis. Mesenchymal cells are known to be required for epithelial cell proliferation. However, the contribution of mesenchymal cells to thymic epithelial cell differentiation is still unclear. In the present study, we have investigated the roles of mesenchymal cells in functional development of epithelial cells in the thymus anlage in patch (ph) mutant mice, which have a primarily defect in mesenchymal cells caused by the absence of platelet-derived growth factor receptor α expression. In the ph/ph thymus anlage, T cell progenitors migrate normally among the epithelial cells, however, they are severely impaired to proliferate and differentiate to CD25-positive cells. Epithelial cells of the ph/ph thymus anlage show severely impaired proliferation and expression of functional molecules, such as SCF, Delta-like 4 and MHC class II, which have crucial roles in T cell development. Moreover, the cultured ph/ph thymus anlage fails to develop into a mature organ supporting full T cell development. Addition of intact thymic mesenchymal cells to organ culture induces development of the ph/ph thymus anlage. In the cultured lobes, added mesenchymal cells contribute to form not only the capsule but also the meshwork structure mingled with epithelial cells. Our present results strongly suggest the roles of mesenchymal cells in functional development of epithelial cells in thymus organogenesis. In addition, our data suggest that mesenchymal cells are required to create the thymic microenvironment and to maintain epithelial architecture and function.