Brain capillary endothelial cells (BCECs) assist in controlling the central nervous system by tightly regulating the passage of molecules between the blood and the brain, and have been termed the blood-brain barrier (BBB). In vitro models of the BBB typically lack the impermeability observed in vivo, which partially stems from removing BCECs from their in vivo microenvironment where perivascular cells such as astrocytes and neurons provide differentiation cues. When cultured in the Petri dish, it is well known that BCECs rapidly de-differentiate in vitro, but questions regarding the magnitude and extent of de-differentiation abound. Thus, genomics techniques were used to assess this complex biological system, and to determine the consequences of transferring BCECs from the brain environment into the petri dish. Suppression subtractive hybridization (SSH) was utilized to compare gene expression levels for intact capillaries (in vivo BBB) with those grown in cell culture (in vitro BBB). Sequencing of SSH products yielded twenty-six genes whose expression levels had changed as a result of culturing. Examples of genes whose expression is downregulated in vitro include organic anion transporter 14 (Oatp14), a brain specific transporter responsible for the passage of thyroid hormones, and carboxypeptidase E (CPE), an enzyme involved in the post-translational processing of neuropeptides. Other genes are upregulated in vitro, such as serine (or cysteine) proteinase inhibitor (Serpine1) that mediates the inhibition of fibrinolysis, and chemokine (C-C motif) ligand 7 (Ccl7), a chemotactic factor that attracts monocytes and eosinophils. These genes represent a variety of cellular functions and include many genes whose levels were previously not known to change. The differentially expressed genes were then used as a “fingerprint” that could be used to assess in vitro model quality upon reintroduction of components normally found in the brain microenvironment. The effects of BCEC culture purity as well as astrocyte and neuron co-culture on in vitro model quality were assessed by quantitative PCR and the resultant genetic fingerprint compared with that found in vivo. These approaches led to a better understanding of the role that the multicellular brain microenvironment plays in conferring BBB properties.