Human periodontal ligament stem cells (hPDLSCs) are promising for periodontal regeneration. However, to date, there has been no report of hPDLSC differentiation into the fibrogenic lineage. There has been no report demonstrating hPDLSC differentiation into all three (osteogenic, fibrogenic and cementogenic fibrogenic) lineages in the same report. The objectives of this study were to harvest hPDLSCs from the periodontal ligaments (PDL) of the extracted human teeth, and use the same vial of hPDLSCs to differentiate into all three (osteogenic, fibrogenic and cementogenic) lineages for the first time. hPDLSCs were harvested from PDL tissues of the extracted premolars. The ability of hPDLSCs to form bone, cementum and collagen fibers was tested in culture mediums. Gene expressions were analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). Immunofluorescence, alizarin red (ARS), Xylenol orange, picro sirius red staining (PSRS), alcian blue staining (ABS) and alkaline phosphatase (ALP) staining were evaluated. In osteogenic medium, hPDLSCs had high expressions of osteogenic genes (RUNX2, ALP, OPN and COL1) at 14 and 21 days (15-20 folds of that of control), and produced mineral nodules and ALP activity (5 and 10 folds those of the control). hPDLSCs in fibrogenic medium expressed high levels of PDL fibrogenic genes (COL1, COL3, FSP-1, PLAP-1 and Elastin) at 28 days (20-70 folds of control). They were stained strongly with F-actin and fibronection, and secreted PDL collagen fibers (5 folds of control). hPDLSCs in cementogenic medium showed high expressions of cementum genes (CAP, CEMP1 and BSP) at 21 days (10-15 folds of control) and synthesized mineralized cementum (50 folds via ABS, and 40 folds via ALP staining, compared to those of control). hPDLSCs differentiated into bone-, fiber- and cementum-forming cells, with potential for regeneration of periodontium to form the bone-PDL-cementum complex. Copyright © 2019 Elsevier Ltd. All rights reserved.