The mammalian cerebral cortex is characterized by its organization into anatomically and functionally discrete regions. During cortical development, a homogeneous-appearing population of cells along the ventricular surface generates the neurons and glia that ultimately form these cytoarchitectonic areas. The limbic system-associated membrane protein (LAMP) is a neuronal, cell surface glycoprotein that identifies neurons restricted to limbic cerebral cortical areas (Levitt, 1984). LAMP is expressed early in development (Horton and Levitt, 1988), and transplantation studies in the rat suggest that cells in the cerebral wall are committed to a limbic or nonlimbic molecular phenotype by embryonic day 14 (E14) (Barbe and Levitt, 1991). However, at E12, cells destined for the cerebral cortex are still multipotential and presumably depend on local, extrinsic signals to adopt a limbic phenotype. We have developed an in vitro assay system for examining the fate of these multipotential progenitors and identifying potential environmental regulators of neuronal differentiation. Regions of the lateral (limbic) and dorsal (nonlimbic) cerebral wall at E12 are dissected, dissociated, and grown in low-density cultures in defined medium. The cells are examined by immunocytochemistry for expression of MAP2, a neuronal cytoskeletal protein, and LAMP to define neuronal differentiation and the expression of a limbic molecular phenotype, respectively. We find that after 4 d in culture, up to 75% of the progenitor cells from presumptive limbic cortex express LAMP upon differentiation. In contrast, only 20-30% of the differentiated cells from presumptive sensorimotor cortex express LAMP. Thus, most cortical progenitors are fated to a limbic or nonlimbic phenotype early in development, and the decision by neuronal stem cells to differentiate into neurons exhibiting this molecular phenotype occurs prior to the completion of neurogenesis, in the absence of subcortical environmental cues.