Brown adipose tissue (BAT) is a thermoregulatory fat with energy-consuming properties. The location and heterogeneity of this tissue makes it complicated to sample before and after interventions in humans, and an in vitro model for mechanistic and molecular studies is therefore of great value. We here describe a protocol for isolation of progenitors from the stromal vascular fraction of BAT biopsies obtained surgically from adult humans. We further present how these cells are differentiated in vitro and finally how they are characterized for thermogenic capacity. Methods for characterization described here include norepinephrine-induced thermogenic gene expression using qPCR; norepinephrine-induced mitochondrial uncoupling using the Seahorse XFe96 Analyzer, and norepinephrine-induced expression of UCP1 using the RNAscope® Technology.