Pericytes and vascular smooth muscle cells, collectively termed mural cells, regulate blood vessel formation, stability, and tone. In the central nervous system, mural cells also regulate the blood-brain barrier (BBB) and mediate neurovascular coupling, among other functions. In vitro cultures of brain mural cells have been incorporated into BBB models and used to study mural cell function and dysfunction. Generating brain mural cells from human pluripotent stem cells (hPSCs) offers a system for examining human mural cell differentiation and disease mechanisms. In this chapter, we describe a serum-free method to differentiate brain mural cells from hPSCs, using lentiviral overexpression of the Notch3 intracellular domain (N3ICD) in a neural crest intermediate. Importantly, the resulting cells express markers characteristic of definitive mural cell identity, including HEYL, TBX2, FOXS1, PDGFRB, RGS5, KCNJ8, and endogenous NOTCH3, and should be useful for a variety of in vitro mural cell modeling applications.