The brain is a main center for control over the other organs in all vertebrate. It is responsible for higher-order functions like processing environmental stimuli or language and it is divided in two hemispheres. In order to achieve such tasks, the right and left sides need to be connected and the corpus callosum is the major bridge (over 200 mio. of axons) channeling and coordinating information between these two hemispheres. Previous works have shown that glial cells (non-neuronal brain cells) found at the midline of the brain are essential for guiding the projections from one side to the other side. We found that two populations of neuronal cells are also required for the proper formation of the corpus callosum and for the guidance of interhemispheric axons during embryonic development. Similar mechanisms could occur in humans and observations of human pathologies with defects in corpus callosum formation suggest that these neurons could be involved. This work thus gives new insights into how neurons together with their glial partners have the ability to shape the corpus callosum, a potential process for guidance in other brain regions.