AbstractDuring early vertebrate forebrain development, pioneer axons establish a symmetrical scaffold descending longitudinally through the rostral forebrain, thus forming the tract of the postoptic commissure (TPOC). In mouse embryos, this tract begins to appear at embryonic day 9.5 (E9.5) as a bundle of axons tightly constrained at a specific dorsoventral level. We have characterized the participation of the Slit chemorepellants and their Robo receptors in the control of TPOC axon projection. In E9.5–E11.5 mouse embryos, Robo1 and Robo2 are expressed in the nucleus origin of the TPOC (nTPOC), and Slit expression domains flank the TPOC trajectory. These findings suggested that these proteins are important factors in the dorsoventral positioning of the TPOC axons. Consistently with this role, Slit2 inhibited TPOC axon growth in collagen gel cultures, and interfering with Robo function in cultured embryos induced projection errors in TPOC axons. Moreover, absence of both Slit1 and Slit2 or Robo1 and Robo2 in mutant mouse embryos revealed aberrant TPOC trajectories, resulting in abnormal spreading of the tract and misprojections into both ventral and dorsal tissues. These results reveal that Slit‐Robo signaling regulates the dorsoventral position of this pioneer tract in the developing forebrain. © 2011 Wiley‐Liss, Inc.