Chronic sublethal hypoxia induces brain adaptations associated with changes in neurovascular behavior. Changes to the neurovasculature also influence the formation of the brain-blood barrier (BBB). In this study, I investigated the influence of chronic sublethal hypoxia on astrocytes, using the coculture transwell model of primary cultured astrocytes and RBE4 (brain endothelial) cells. Using a 3D collagen gel model, cytoplasmic processes of astrocytes extended to clumps of endothelial cells. The numbers of astrocytes increased in cocultured and chronic hypoxic environments in the transwell model. Western blotting showed increased production of glial fibrillar acidic protein (GFAP) and proliferating cellular nuclear antigen (PCNA) in chronic hypoxia. I also confirmed the influence of hypoxia on the behavior of astrocytes in this model, using confocal microscopy. The numbers of cytoplasmic processes of astrocytes within the membrane increased in z sections. These data support the idea that chronic hypoxia might induce alterations in the formation of the BBB as part of the adaptation of the brain to chronic hypoxia. These transwell and 3D collagen gel models will probably be useful for functional as well as morphological experiments.