How do neurons in the medial entorhinal cortex (MEC) organize themselves during development to establish networks involved in complex functions like learning and memory? From developmental studies of cortical and subcortical structures, one transcription factor called Bcl11b (encoding Ctip2) is responsible for neuronal differentiation and axonal pathfinding. Interestingly, in the MEC, Bcl11b is expressed by Reelin-positive (Reln+) projection neurons in layers 2 and by intracortical projection neurons in layer 5a . Because Bcl11b is widely expressed by these early born MEC neurons, we hypothesized that Bcl11b is critical to MEC development. Thus, we knocked out Bcl11b from all telencephalic glutamatergic neurons using the Emx1-Cre mouse line and performed immunohistochemistry in MEC at postnatal days 6 and 30. Strikingly, we observed a change in Reln+ cell number in layer 2 and an overall change in distribution of neuronal subtype across deep and superficial layers of the MEC at both timepoints. We then microdissected the MEC from 6-day-old mice and performed snRNA-seq to corroborate cell type changes and identify gene network disturbances due to Bcl11b knockout. Our analysis showed both loss and gain of cell types, an increase in superficial layer neurons and a decrease in deep layers neurons. Further analysis of sn-mRNA expression revealed that Reln+ neurons exhibited gene network changes in the Bcl11b knockout mice related to dendritic and axonal assembly. Our current results suggest that Bcl11b is a crucial genetic driver of cytoarchitectural establishment in the developing MEC.