To date, little is published on the characterization and therapeutic potential of human mesenchymal stromal cells (MSCs) derived from hair follicle dermal sheath (DS). We present protocols for the isolation and culture of human DS-MSCs starting with the use of a dissecting microscope to separate out dermal sheaths from hair follicles for trypsin digestion. We also present the protocols for the adipogenic, osteogenic, and chondrogenic differentiation of these DS-MSCs as we seek to harness these cells for potential applications in stem cell therapy and tissue engineering.