Immune checkpoint inhibitors improved the survival rate of patients with unresectable melanoma. However, some patients do not respond, and variable immune-related adverse events have been reported. Therefore, more effective and antigen-specific immune therapies are urgently needed. We previously reported the efficacy of an immune cell therapy with immortalized myeloid cells derived from induced pluripotent stem cells (iPS-ML). In this study, we generated OX40L-overexpressing iPS-ML (iPS-ML-Zsgreen-OX40L) and investigated their characteristics and in vivo efficacy against mouse melanoma. We found that iPS-ML-Zsgreen-OX40L suppressed the progression of B16-BL6 melanoma, and prolonged survival of mice with ovalbumin (OVA)-expressing B16 melanoma (MO4). The number of antigen-specific CD8 T cells was higher in spleen cells treated with OVA-peptide-pulsed iPS-ML-Zsgreen-OX40L than in those without OX40L. The OVA-peptide-pulsed iPS-ML-Zsgreen-OX40L significantly increased the number of tumor-infiltrating T lymphocytes (TILs) in MO4 tumor. Flow-cytometry showed decreased regulatory T cells but increased effector and effector memory T cells among the TILs. Although we plan to use allogeneic iPS-ML in the clinical applications, iPS-ML showed the tumorgenicity in the syngeneic mice model. Incorporating the suicide gene is necessary to ensure the safety in the future study. Collectively, these results indicate that iPS-ML-Zsgreen-OX40L therapy might be a new method for antigen-specific cancer immunotherapy.This article is protected by copyright. All rights reserved.