To avoid heterogeneous responses when activating TLR signaling in CLL cells, we used a mix of TLR agonists that stimulates TLR1, TLR2, TLR6, and TLR9, and showed that it reliably triggered activation of the NF-κB and JAK-STAT signaling pathways, secretion of cytokines, and enhanced CLL cell migration and proliferation in vitro in all CLL samples, highlighting the relevance of TLR signaling in CLL pathobiology. Here, TLR9 is linked to B-cell chronic lymphocytic leukemia.