Previous work has demonstrated that fusion cells generated from autologous monocyte-derived dendritic cells (MoDCs) and whole tumor cells induce efficient antigen-specific cytotoxic T lymphocytes. A major limitation to the use of this strategy is the availability of adequate amounts of autologous tumor cells. Moreover, MoDCs from cancer patients are often defective in their antigen-processing and presentation machinery. In this study, two types of allogeneic cells, a leukemia plasmacytoid dendritic cell (pDC) line (PMDC05) and pancreatic cancer cell lines (PANC-1 or MIA PaCa-2), were fused instead of autologous MoDCs and tumor cells. We created four types of pDC/tumor fusion cells by alternating fusion partners and treating with lipopolysaccharide (LPS): i) PMDC05 fused with PANC-1 (pDC/PANC-1), ii) PMDC05 fused with MIA PaCa-2 (pDC/MIA PaCa-2), iii) LPS-stimulated pDC/PANC-1 (LPS-pDC/PANC-1) and iv) LPS-stimulated pDC/MIA PaCa-2 (LPS-pDC/MIA PaCa-2) and examined their antitumor immune responses. The LPS-pDC/tumor cell fusions were the most active, as demonstrated by their: i) upregulated expression of HLA-DR and CD86 on a per-fusion-cell basis, ii) increased production of IL-12p70, iii) generation of a higher percentage of IFN-γ-producing CD4⁺ and CD8⁺ T cells and iv) augmented induction of MUC1-specific CD8⁺ T cells that lyse target tumor cells. This study provides the first evidence for an in vitro induction of antigen-specific cytotoxic T lymphocytes by LPS-stimulated fusion cells generated from leukemia plasmacytoid DCs and tumor cells and suggests that this strategy has potential applicability to the field of adoptive immunotherapy.