The diencephalon is a complex brain area which derives from the caudal region of the prosencephalon. This structure is divided in horizontal zones along the dorso-ventral axis. The mechanisms that govern the formation of this axis during development remain unclear. Different signaling molecules have been described as involved in this process, known as morphogenes, which induce a specific neural fate to the neighbouring epithelium. In order to demonstrate that one of these signaling molecules, Wnt1, has a critical role in the correct establishment of the diencephalic territories, we carried out gain- and loss-of-function experiments. Our results indicate that Wnt1 signal has a key role in the structuration of the pretectal territories during embryonic development, as well as in the correct development of some diencephalic dorsal structures. Moreover, we demonstrate that Wnt1 signal is required for the diencephalic dorso-ventral regionalization. We further show that Wnt1 is directly induced by Bmp4, unraveling the molecular regulatory code modulating this regionalization in the diencephalon.