Millions of people experience consequential tooth loss related to irretrievable damage of the periodontium caused by deep caries, severe periodontal diseases or irreversible trauma, resulting in a decreased quality of life. Hence, dental scientists have focused much attention on tissue engineering techniques in an effort to address this condition. The periodontium is composed mainly of two soft tissues and two hard tissues; the former includes the periodontal ligament (PDL) tissue and gingival tissue, and the latter includes alveolar bone and cementum covering the tooth root. In particular, PDL is a dynamic connective tissue that is subjected to continual adaptation to maintain tissue size and width, as well as structural integrity, including fibers and bone modeling. The main role of PDL is to anchor the tooth root to the alveolar bone socket tightly, cushioning mechanical load that proceeds from mastication. Thus, PDL tissue constitutes the bedrock of periodontium to determine the life-span of tooth. In this chapter, we describe the phenotypes of multipotent clonal human PDL progenitor/stem cell lines that we have recently established, and present a comparison with PDL stem cells (PDLSCs). PDLSCs represent typical properties of bone marrow-derived mesenchymal stem cells (BMMSCs). PDLSCs exhibit a self-renewing capacity and express cell surface markers similar to BMMSCs. PDLSCs also possess the multipotential to differentiate into various types of cells, such as osteoblast-like cells, adipocytes, chondrocytes and neurocytes, in vitro. Additionally, PDLSCs represent a distinctive potential to form cementumand PDL-like tissues in vivo, suggesting that PDLSCs might belong to a unique population of somatic stem cells. Since a very small number of stem cells are included in PDL tissue, researchers have tried to establish immortalized PDL stem cell lines for the convenience and consistency of analyses. Recently, we succeeded in establishing two clonal human PDL cell lines with multipotential by transduction with Simian Virus (SV) 40 large T-antigen and human telomerase reverse transcriptase. These clonal cell lines exhibited unique characteristics: cell line 1-11 demonstrated osteoblastic and adipocytic differentiation capabilities, and cell line 1-17 exhibited the potential to differentiate into osteoblasts, adipocytes, chondrocytes and neurocytes. Flow cytometric analysis demonstrated that the percentage of cells expressing BMMSC markers was very high in both cell lines. On the other hand, cell line 1-17 expressed embryonic stem (ES) cell marker genes at higher levels, compared with cell line 1-11. Furthermore, both cell lines expressed PDL phenotype-related molecules at equivalent levels while BMMSCs showed little expression. When transplanted into PDL defects formed