A novel signal transduction pathway utilized by extracellular HSP70 has been identified. This study demonstrates that HSP70 activates proinflammatory cytokine production through the MyD88/IRAK/NF-κB signaling pathway and utilizes both TLR2 (Gram-positive bacteria receptor) and TLR4 (Gram-negative bacteria receptor) in a CD14-dependent manner. These findings suggest that HSP70 can act as an endogenous ligand for TLRs, potentially linking innate and adaptive immunity. The study also shows that HSP70 synergizes with TLR2 and TLR4 to enhance cytokine production, indicating a role in immune activation. The results highlight the importance of CD14 in HSP70 signaling via TLRs and suggest that HSP70 may serve as a danger signal to the immune system. The study further demonstrates that HSP70 can induce cytokine production and co-stimulatory molecule expression in dendritic cells, enhancing their ability to prime T cells. These findings have implications for the development of pharmacological or molecular tools to modulate HSP70-induced functions in various diseases. The study also addresses concerns about endotoxin contamination, showing that boiling HSP70 abrogates its effects, while LPS remains unaffected. Overall, the study provides new insights into the role of HSP70 in immune signaling and its potential therapeutic applications.A novel signal transduction pathway utilized by extracellular HSP70 has been identified. This study demonstrates that HSP70 activates proinflammatory cytokine production through the MyD88/IRAK/NF-κB signaling pathway and utilizes both TLR2 (Gram-positive bacteria receptor) and TLR4 (Gram-negative bacteria receptor) in a CD14-dependent manner. These findings suggest that HSP70 can act as an endogenous ligand for TLRs, potentially linking innate and adaptive immunity. The study also shows that HSP70 synergizes with TLR2 and TLR4 to enhance cytokine production, indicating a role in immune activation. The results highlight the importance of CD14 in HSP70 signaling via TLRs and suggest that HSP70 may serve as a danger signal to the immune system. The study further demonstrates that HSP70 can induce cytokine production and co-stimulatory molecule expression in dendritic cells, enhancing their ability to prime T cells. These findings have implications for the development of pharmacological or molecular tools to modulate HSP70-induced functions in various diseases. The study also addresses concerns about endotoxin contamination, showing that boiling HSP70 abrogates its effects, while LPS remains unaffected. Overall, the study provides new insights into the role of HSP70 in immune signaling and its potential therapeutic applications.