Growing axons are often guided to their final destination by intermediate targets. In the developing spinal cord and optic nerve, specialized cells at the embryonic midline act as intermediate targets for guiding commissural axons. Here we investigate whether similar intermediate targets may play a role in guiding cortical axons in the developing brain. During the development of the corpus callosum, cortical axons from one cerebral hemisphere cross the midline to reach their targets in the opposite cortical hemisphere. We have identified two early differentiating populations of midline glial cells that may act as intermediate guideposts for callosal axons. The first differentiates directly below the corpus callosum forming a wedge shaped structure (the glial wedge) and the second differentiates directly above the corpus callosum within the indusium griseum. Axons of the corpus callosum avoid both of these populations in vivo. This finding is recapitulated in vitro in three-dimensional collagen gels. In addition, experimental manipulations in organotypic slices show that callosal axons require the presence and correct orientation of these populations to turn toward the midline. We have also identified one possible candidate for this activity because both glial populations express the chemorepellent molecule slit-2, and cortical axons express the slit-2 receptors robo-1 and robo-2. Furthermore, slit-2 repels-suppresses cortical axon growth in three-dimensional collagen gel cocultures.