Significance The mammalian neocortex consists of six histologically distinct layers, called layer 1 (L1) to L6. Each layer contains several subtypes of neurons, which are characterized by specific cell morphology, gene expression profile, and connectivity to other regions of the central nervous system. As these neurons are mostly born from common progenitor cells, it is important to know how progenitor cells acquire a certain cell type during development. Here, we reveal the essential interactions between two types of transcription factors, Brn1/2 and Rorb, for subtype specification. Brn1/2 and Rorb are expressed in L2/3 and L4, respectively, in the mature neocortex. We found that Brn1/2 and Rorb repress each other and that this reciprocal repression is important for L2/3 and L4 specification.