Vascular endothelial cells (ECs) derived from the central nervous system (CNS) variably lose their unique barrier properties during in vitro culture, hindering the development of robust assays for blood-brain barrier (BBB) function, including drug permeability and extrusion assays. In previous work (Sabbagh et al., 2018) we characterized transcriptional and accessible chromatin landscapes of acutely isolated mouse CNS ECs. In this report, we compare transcriptional and accessible chromatin landscapes of acutely isolated mouse CNS ECs versus mouse CNS ECs in short-term in vitro culture. We observe that standard culture conditions are associated with a rapid and selective loss of BBB transcripts and chromatin features, as well as a greatly reduced level of beta-catenin signaling. Interestingly, forced expression of a stabilized derivative of beta-catenin, which in vivo leads to a partial conversion of non-BBB CNS ECs to a BBB-like state, has little or no effect on gene expression or chromatin accessibility in vitro.