AbstractWe have identified two Semaphorin genes, Sema 3D and Sema 3F, as being specifically expressed in the developing parathyroid and thymus. The thymus and parathyroid originate from a common primordium that develops from the third pharyngeal pouch in mice. The expression of Sema 3D and Sema 3F was compared with that of gcm2, Pax1, and Neuropilin‐1 and ‐2, and genes encoding a variety of Plexins by in situ hybridization during mouse pharyngeal development. Sema 3D was specifically expressed in the dorsal and cranial portions of the primordium developing from the third pouch at mouse embryonic day (E) 10.5. That the expression pattern of Sema 3D was consistent with that of gcm2 indicates that Sema 3D was expressed in the parathyroid‐specific domains of the developing third pouch. The parathyroid‐specific pattern of Sema 3D expression continued until E17.5. By contrast, Sema 3F was expressed in both the parathyroid and thymic domains of the common primordium at E10.5, and the expression levels in both tissues were decreased during development. The Semaphorin receptors were expressed in the blood vessels, nerves, and mesenchymal cells adjacent to the common primordium or the separated parathyroid and thymus during development. Our results show that Sema 3D and Sema 3F genes were overlappingly and distinctly expressed in the developing parathyroid and thymus, respectively, and that Semaphorin signaling might play roles in the interactions between these organs and surrounding tissues such as nerves and blood vessels, and in the recruitment of lymphoid cells. Developmental Dynamics 237:1699–1708, 2008. © 2008 Wiley‐Liss, Inc.