Objective: In vitro, the culture media in which human dental pulp stem cells (hDPSCs) are grown are supplemented with specific growth factors that induce cell cycle entry and differentiation. Lysate platelet-rich fibrin (L-PRF) is a unique and stable growth factor supplement produced from platelets lysed by freezing-thawing. In this study, we aimed to analyze the potential effects of L-PRF on hDPSC differentiation. Methods: We divided hDPSCs isolated from human third molars at the second passage into five culture media groups treated with 1%, 5%, 10%, and 25% L-PRF or 10% fetal bovine serum (control). After 7 days, we evaluated hDPSC differentiation using an enzyme-linked immunosorbent assay specific for dentin sialophosphoprotein and Alizarin-Red staining. Results: None of our analyses revealed any significant differences between the L-PRF-and control-treated cells. Conclusion: L-PRF could potentially induce the differentiation of hDPSCs in vitro. © 2020 The Authors.