The thymus harbors an organ-typical dense network of branching and anastomosing blood vessels. To address the molecular basis for morphogenesis of this thymus-specific vascular pattern, we have inactivated a key vascular growth factor, VEGF-A, in thymus epithelial cells (TECs). Both Vegf-A alleles were deleted in TECs by a complementation strategy termed nude mouse [mutated in the transcription factor Foxn1 (forkhead box N1)] blastocyst complementation. Injection of Foxn1(+/+) ES cells into Foxn1(nu/nu) blastocysts reconstituted a functional thymus. By dissecting thymus stromal cell subsets, we have defined, in addition to medullary TECs (mTECs) and cortical TECs (cTECs), another prominent stromal cell subset designated cortical mesenchymal cells (cMes). In chimeric thymi, mTECs and cTECs but not cMes were exclusively ES cell-derived. According to this distinct origin, the Vegf-A gene was deleted in mTECs and cTECs, whereas cMes still expressed Vegf-A. This genetic mosaic was associated with hypovascularization and disruption of the organ-typical network of vascular arcades. Thus, vascular growth factor production by TECs is required for normal thymus vascular architecture. These experiments provide insights into Foxn1-dependent and Foxn1-independent stromal cell development and demonstrate the value of this chimeric approach to analyzing gene function in thymus epithelium.