Homeostasis of the central nervous system (CNS) microenvironment is essential for its normal function and is maintained by the blood-brain barrier (BBB). The BBB proper is made up of endothelial cells (ECs) interconnected by tight junctions (TJs) that reveal a unique morphology and biochemical composition of the body’s vasculature. In this article, we focus on developmental aspects of the BBB and describe morphological as well as molecular special features of the neurovascular unit (NVU) involved in barrier induction. Recently, we and others identified the Wnt/β-catenin pathway as crucial for brain angiogenesis, TJ and BBB formation. Based on these findings we discuss other pathways and molecular interactions for BBB establishment and maintenance. At the morphological level, our concept favors a major role for polarized astrocytes (ACs) therein. Orthogonal arrays of particles (OAPs) that are the morphological correlate of the water channel protein aquaporin-4 (AQP4) are specifically formed in the membrane of the AC endfoot. The polarized AC endfoot and hence OAPs are dependent on agrin and dystroglycan, of which agrin is a developmentally regulated extracellular matrix (ECM) component. Understanding the mechanisms leading to BBB development will be key to the understanding of barrier maintenance that is crucial for, but frequently disturbed, in the diseased adult brain.