The integrity of the fluid–blood barrier in the stria vascularis is critical for maintaining inner ear homeostasis, especially for sustaining the endocochlear potential, an essential driving force for hearing function. However, the mechanisms that control intrastrial fluid–blood barrier permeability remain largely unknown. At the cellular level, the intrastrial fluid–blood barrier comprises cochlear microvascular endothelial cells connected to each other by tight junctions (TJs), an underlying basement membrane, and a second line of support consisting of cochlear pericytes and perivascular resident macrophage-type melanocytes. In this study, we use a newly established primary cell culture-based in vitro model to show that endothelial cells, pericytes, and perivascular resident macrophage-type melanocytes interact to control intrastrial fluid–blood barrier permeability. When the endothelial cell monolayer was treated with pericyte—or perivascular resident macrophage-type melanocyte—conditioned media, the permeability of the endothelial cell monolayer was significantly reduced relative to an untreated endothelial cell monolayer. Further study has shown the pericytes and perivascular resident macrophage-type melanocytes to regulate TJ expression in the endothelial cell monolayer. The new cell culture-based in vitro model offers a unique opportunity to obtain information on the organ-specific characteristics of the cochlear blood/tissue barrier. Our finding demonstrates the importance of signaling among pericytes, endothelial cells, and perivascular resident macrophage-type melanocytes to the integrity of the intrastrial fluid–blood barrier.