Vascular assembly, patterning, and maintenance is a complex and highly regulated process that begins with the formation of a primary capillary plexus by means of angiogenesis or vasculogenesis and ends when the primitive vessels have been remodeled into quiescent, differentiated vessels. Differentiated or "mature" microvessels are characterized in large part by their association with pericytes, and failure of these interactions results in severe, and often lethal, defects that have been implicated in a number of human pathologic conditions, including tumor angiogenesis, diabetic microangiopathy, ectopic tissue calcification, stroke, and dementia. This chapter describes methods that can be used to isolate and culture primary pericytes, as well as to study pericyte-endothelial cell interactions with in vitro cell culture systems.