The retinal pigment epithelium is an important barrier to drug transport as well as contributing to the normal functioning of the photoreceptors. The contributions of glial cells in the retina to the maintenance and development of this barrier is important. There is evidence that retinal secreted factors play a role in the induction and maintenance of the outer blood retinal barrier. One possible source of such factors are the retinal glial cells, astrocytes and Müller cells, which may influence tight junction formation and maturation. The aim of this study was to examine the changes in the trans-epithelial resistance (TER), as a measure of barrier integrity, on cell lines of epithelial origin (ECV304 and ARPE-19) following co-culture with glial cell lines (C6 and MIO-M1) or with the addition of medium conditioned by these cells. One cell line, ECV304, showed a significant increase in the TER in response to glial secreted factors whilst ARPE-19 did not. This finding suggests that ECV304 responds well to glial factors and may be useful for further studies of the factors that affect tight junction formation through glial cell induction in vitro.