Innate immune cells recognize pathogens through pattern recognition receptors (PRRs), and activation of PRRs induces downstream signaling pathways to mount appropriate immune responses. Pathogens usually carry multiple ligands, which can simultaneously activate multiple PRRs. The cooperation of multiple PRRs and consequential crosstalk between their downstream pathways could enhance cytokine expression, which is required for effective immune responses. On the other hand, immune over-activation could also harm the host if immune homeostasis is not restored. Therefore, it is important to understand the mechanisms of PRR cooperation during an infection. As the best characterized PRRs, Toll-like receptors (TLRs) have an important role in pathogen recognition, and crosstalk among TLRs is common. In this review, we provide an update on the recent findings on the mechanisms of TLR cooperation. We summarize the known mechanisms and provide a future perspective on TLR crosstalk study, with a caution against the use of multiple TLR ligands as adjuvants in therapeutic strategies.