Hippocampus-associated genes that orchestrate the formation of the compact stratum pyramidale are largely unknown. The BTB (broad complex, tramtrack,bric-a-brac)-zinc finger gene Zbtb20 (also known as HOF, Znf288,Zfp288) encodes two protein isoforms, designated Zbtb20S and Zbtb20L, which are expressed in newborn pyramidal neurons of the presumptive hippocampus in mice. Here, we have generated transgenic mice with ectopic expression of Zbtb20S and Zbtb20L in immature pyramidal neurons differentiated from multipotent non-hippocampal precursors. The subiculum and posterior retrosplenial areas in these mice were transformed into a three-layered hippocampus-like cortex with a compact homogenous pyramidal cell layer. Severe malformations of lamination occur in neocortical areas, which coincide with a deficiency in expression of cortical lamination markers. The alterations in cortical cytoarchitecture result in behavioral abnormalities suggestive of a deficient processing of visual and spatial memory cues in the cerebral cortex of adult Zbtb20 transgenic mice. Overall,our in vivo data suggest that Zbtb20 functions as a molecular switch for a pathway that induces invariant pyramidal neuron morphogenesis and suppression of cell fate transitions in newborn neurons.