Mouse retinal ganglion cell axons growing from the eye encounter embryonic neurons at the future site of the optic chiasm. After in vivo ablation of these chiasm neurons with a monoclonal antibody and complement, retinal axons did not cross the midline and stalled at approximately the entry site into the chiasm region. Thus, in the mouse, the presence of early-generated neurons that reside at the site of the future chiasm is required for formation of the optic chiasm by retinal ganglion cell axons.