Dendritic cells (DC) have a unique capacity to present external antigens to CD8+ T cells, i.e. cross-presentation. However, it is not fully established whether the ability to cross-presentation is restricted to a unique subset of DC in humans. Here, we show that two major myeloid DC subsets, i.e. Langerhans cells (LC) and interstitial DC (Int-DC), have the ability to cross-present antigens to CD8+ T cells in vitro. LC and Int-DC were obtained from DC generated by culturing human CD34+-hematopoietic progenitor cells with GM-CSF, FLT3-L, and TNF-α (CD34-DC). Both DC subsets were able to capture necrotic/apoptotic allogeneic melanoma cells and present antigens to CD8+ T cells, resulting in efficient priming of naive CD8+ T cells into CTL capable of killing melanoma cells. Strikingly, a single stimulation with either subset (LC or Int-DC) or total CD34-DC loaded with necrotic/apoptotic melanoma cells was sufficient to activate melanoma-specific memory CD8+ T cells obtained from patients with metastatic melanoma to become effective CTL. Thus, this study provides the rationale to use CD34-DC loaded with necrotic/apoptotic allogeneic melanoma cells in a clinical trial.