Human periodontal ligament stem cells (PDLSCs) are a unique population of mesenchymal stem cells (MSCs) that demonstrate the capacity to generate cementum- and periodontal ligament-like structures in vivo. As such, PDLSCs represent a promising cell-based therapy in reconstructive dentistry for the treatment of periodontal disease. The present chapter describes two methods for isolating PDLSCs from human PDL tissue including traditional plastic adherence, and immunomagnetic selection based on the expression of MSC-associated surface markers STRO-1 antigen, CD146 (MUC-18), CD29 (Integrin β-1), CD44, and CD106 (VCAM-1). Although no single antibody demonstrates specificity for MSCs, isolation based on expression of individual markers results in homogenous preparations of PDLSCs. Methods to further characterize the immunophenotype and multipotent capacity of PDLSCs to differentiate into adipocytes, osteoblast-, and cementoblast-like cells in vitro, and cementum- and periodontal ligament-like tissues in vivo, are also described.