The Drosophila optic lobe comprises a wide variety of neurons forming laminar and columnar structures similar to the mammalian brain. The Drosophila optic lobe may provide an excellent model to investigate various processes of brain development. However, it is poorly understood how neuronal specification is regulated in the optic lobe to form a complicated structure. Here we show that the Brain-specific-homeobox (Bsh) protein, which is expressed in the lamina and medulla ganglia, is involved in specifying neuronal identity. Bsh is expressed in L4 and L5 lamina neurons and in Mi1 medulla neurons. Analyses of loss-of-function and gain-of-function clones suggest that Bsh is required and largely sufficient for Mi1 specification in the medulla and L4 specification in the lamina. Additionally, Bsh is at least required for L5 specification. In the absence of Bsh, L5 is transformed into glial cells.