Cholangiocarcinoma (CCA) cells paradoxically express the death ligand, tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL) and, therefore, are dependent upon potent survival signals to circumvent TRAIL cytotoxicity. CCAs are also highly desmoplastic cancers with a tumor microenvironment rich in myofibroblasts (MFBs). Herein, we examine a role for MFB‐derived CCA survival signals. We employed human KMCH‐1, KMBC, HuCCT‐1, TFK‐1, and Mz‐ChA‐1 CCA cells, as well as human primary hepatic stellate and myofibroblastic LX‐2 cells, for these studies. In vivo experiments were conducted using a syngeneic rat orthotopic CCA model. Coculturing CCA cells with myofibroblastic human primary hepatic stellate cells or LX‐2 cells significantly decreased TRAIL‐induced apoptosis in CCA cells, a cytoprotective effect abrogated by neutralizing platelet‐derived growth factor (PDGF)‐BB antiserum. Cytoprotection by PDGF‐BB was dependent upon Hedgehog (Hh) signaling, because it was abolished by the smoothened (SMO; the transducer of Hh signaling) inhibitor, cyclopamine. PDGF‐BB induced cyclic adenosine monophosphate–dependent protein kinase–dependent trafficking of SMO to the plasma membrane, resulting in glioma‐associated oncogene (GLI)2 nuclear translocation and activation of a consensus GLI reporter gene‐based luciferase assay. A genome‐wide messenger RNA expression analysis identified 67 target genes to be commonly up‐ (50 genes) or down‐regulated (17 genes) by both Sonic hedgehog and PDGF‐BB in a cyclopamine‐dependent manner in CCA cells. Finally, in a rodent CCA in vivo model, cyclopamine administration increased apoptosis in CCA cells, resulting in tumor suppression. Conclusions: MFB‐derived PDGF‐BB protects CCA cells from TRAIL cytotoxicity by a Hh‐signaling–dependent process. These results have therapeutical implications for the treatment of human CCA. (HEPATOLOGY 2011;)