Priming of CD8+T cells requires two signals, one produced by T‐cell receptor recognition of antigen, and a second that is often provided by the innate immune response. In this context, antigens non‐covalently or covalently associated with heat shock proteins (HSP) are internalized and processed in antigen‐presenting cells (APC) to be presented by MHC I molecules to CD8+T cells, thus, signal 1 has been well characterized in this pathway of cross‐presentation. Signal 2 is not fully understood, although there are reports that Toll‐like receptors (TLRs) interact with HSP and activate APC. The ability of HSP to activate APC through TLRs is, however, controversial because of the possibility of endotoxin contamination. Using a variety of TLR KO mice, we present evidence that TLRs (TLR2, 3, 4, 7, and 9) and their adaptor molecules MyD88 and IRAK4 are dispensable in cross‐priming by a mycobacterial HSP70–antigen (ovalbumin as a model antigen) fusion protein; in contrast, MyD88/IRAK4, but not TLRs, are required for tumor rejection induced by the same reagent. Our results indicate that HSP‐mediated cross‐priming uses a second signal produced by mechanisms other than TLR cascades. We hypothesize that efficient cross‐priming by HSP70 alone is insufficient for tumor rejection and that MyD88/IRAK4‐dependent inflammatory stimulation, which might contribute to maintenance of the initially primed effector cells, is required to eradicate tumor burden. (Cancer Sci 2012; 103: 851–859)