Tendon and ligament injuries represent highly prevalent and unmet clinical challenge that may significantly benefit from tissue engineering therapeutic strategies, once optimal cell source and biomolecules regulating tendon homeostasis are properly defined. Herein, we aimed to evaluate the expression of tendon/ligament markers in two novel cell populations, namely human dental pulp stem cells (DPSCs) and periodontal ligament cells (PDLCs), in response to supplementation with TGF-β ligands relevant for tendon development and healing, as well as under standard tri-lineage differentiation conditions. DPSCs and PDLCs were isolated from sound human permanent molars removed for orthodontic reasons. Pulp tissue and periodontal ligament were minced and digested with collagenase (3mg/mL) and cells were expanded in α-MEM supplemented with 10% fetal bovine serum (basal medium). To evaluate the susceptibility of DPSCs and PDLCs to tenogenic induction, cells were seeded at density of 1000 cells/cm² and cultured up to 21 days in basal medium or media supplemented with TGF-β3 (10ng/ml), or GDF-5 (50 ng/ml). Cell response was evaluated weakly by analysis of expression of tendon, bone and cartilage markers, employing real time RT-PCR and immunocytochemistry. A significant increase in collagen I and collagen III expression was observed with the culture progression in all conditions, with abundant matrix being deposited by day 14. A significant upregulation of scleraxis expression was demonstrated in response to supplementation with TGF-β3 in both cell populations, when compared to basal medium and medium with GDF-5. It was concluded that TGF-β3 may represent an effective inducer of stem cell tenogenic differentiation.