Background <p>Human dental pulp stem cells (hDPSCs) are oral-derived mesenchymal stem cells with high proliferative capacity and odontogenic differentiation potential, making them relevant for dental pulp and dentin–pulp complex regeneration. This in vitro study evaluated the effects of collagen membrane and mineral trioxide aggregate (MTA) conditioned media, individually and in combination, on hDPSC viability, odontogenic differentiation, and mineralization.</p> Methods <p>hDPSCs were isolated, expanded, and characterized by flow cytometry, then cultured in osteogenic differentiation medium and treated with conditioned media derived from collagen membrane, MTA, or their combination. Cell viability was assessed after 48&#xa0;h using the MTT assay. Odontogenic differentiation was evaluated after 7 days by alkaline phosphatase activity and Alizarin Red S staining, while late-stage differentiation was analyzed after 21 days by quantitative real-time PCR of odontogenic-associated genes (<i>RUNX2</i>, <i>DMP1</i>, and <i>BMP2</i>).</p> Results <p>Collagen membrane–conditioned medium moderately increased cell viability but showed limited effects on odontogenic differentiation. In contrast, MTA-conditioned medium significantly enhanced differentiation and mineral deposition. The combined collagen membrane + MTA–conditioned medium produced the most pronounced effects, significantly increasing alkaline phosphatase activity, odontogenic gene expression, and mineralized matrix formation.</p> Conclusion <p>These findings suggest that soluble factors released from collagen membrane and MTA act synergistically to promote odontogenic differentiation of hDPSCs in vitro, with potential relevance to dental pulp regenerative strategies.</p> Graphical Abstract <p></p>