We have previously observed that TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) induces acquired TRAIL resistance by increasing Akt phosphorylation and Bcl-xL expression. In this study, we report that Src, c-Cbl, and PI3K are involved in the phosphorylation of Akt during TRAIL treatment. Data from immunoprecipitation and immunoblotting assay reveal that Src interacts with c-Cbl and PI3K. Data from immune complex kinase assay demonstrate that Src can directly phosphorylate c-Cbl and PI3K p85 subunit protein. Data from gene knockdown experiments with an RNA interference (RNAi) technique show that c-Cbl is involved in the interaction between Src and PI3K p85 during TRAIL treatment, playing an important role in TRAIL-induced Akt phosphorylation. Taken together, c-Cbl may act as a mediator to regulate the Src-PI3K-Akt signal transduction pathway during TRAIL treatment.