The dentate gyrus of rodents is characterized by a highly laminar organization: above a compact granule cell layer, commissural/associational (C/A) fibers terminate on proximal granule cell dendrites and entorhinal fibers terminate on distal granule cell dendrites in a nonoverlapping manner. To gain insights into mechanisms that underlie the formation of this laminar structure, we studied mice deficient for BETA2/NeuroD, a basic helix-loop-helix transcription factor essential for granule cell differentiation. Anterograde tracing was used to label C/A and entorhinal fibers and combined with confocal double immunofluorescence for calbindin, calretinin, parvalbumin, and reelin to visualize putative target cells. The dentate gyrus of mutant mice contained only few granule cells, which formed a cap-like structure adjacent to area CA3. Despite the severe hypoplasia of the dentate gyrus, the remaining BETA2/NeuroD-deficient granule cells expressed mature markers, extended dendrites into the molecular layer, and extended mossy fibers into area CA3. Entorhinal and C/A fibers terminated in a nonoverlapping manner in the dendritic field overlying the rudiment. Entorhinal fibers terminated in the outermost portion of the dentate gyrus where they surrounded reelin-positive Cajal-Retzius cells, and C/A fibers terminated above and within the dentate rudiment. The laminar termination of C/A fibers was closest to normal in zones of the rudiment in which granule cells were densely packed. These data indicate that granule cells are able to differentiate in the absence of BETA2/NeuroD and suggest that the signals underlying the laminar anatomy of the dentate gyrus are present in the absence of most target cells.