The optic tectum is composed of multiple layers, which are formed by radial and tangential migration during development. We found that NRP1 (Neuropilin 1)-mediated Sema3A signals are involved in the process of tectal laminar formation, which is elaborated by tangential migration. In the developing chick tectum, NRP1, a receptor for Sema3A, is expressed in MAP2 (microtubule-associated protein 2)-positive intermediate layers IV/V. Sema3A itself is a diffusible guidance factor and is expressed in the overlying layer VI. By stable fluorescent labeling of tectal cells, we show that MAP2-positive intermediate layers are formed by the neurons that have been dispersed by tangential migration along the tectal efferent axons. When Sema3A was misexpressed during laminar formation, local Sema3A repelled the tangential migrants, thus eliminating MAP2-positive neurons that express NRP1. Furthermore, in the absence of the MAP2-positive neurons, tectal layers were disorganized into an undulated form, indicating that MAP2-positive intermediate layers are required for proper laminar formation. These results suggested that NRP1-mediated Sema3A signals provide repulsive signals for MAP2-positive neurons to segregate tectal layers, which is important to coordinate laminar organization of the optic tectum.