Abstract Common infections have long been proposed to play a role in the development of pediatric B-cell acute lymphoblastic leukemia (B-ALL). However, epidemiologic studies report contradictory effects of infection exposure on subsequent B-ALL risk, and no specific pathogen has been definitively linked to the disease. A unifying mechanism to explain the divergent outcomes could inform disease prevention strategies. We previously reported that the pattern recognition receptor (PRR) ligand Poly(I:C) exerted effects on B-ALL cells that were distinct from those observed with other nucleic acid–based PRR ligands. Here, using multiple double-stranded RNA (dsRNA) moieties, we show that the overall outcome of exposure to Poly(I:C) reflects the balance of opposing responses induced by its ligation to endosomal and cytoplasmic receptors. This PRR response biology is shared between mouse and human B-ALL and can increase leukemia-initiating cell burden in vivo during the preleukemia phase of B-ALL, primarily through tumor necrosis factor α signaling. The age of the responding immune system further influences the impact of dsRNA exposure on B-ALL cells in both mouse and human settings. Overall, our study demonstrates that potentially proleukemic and antileukemic effects can each be generated by the stimulation of pathogen recognition pathways and indicates a mechanistic explanation for the contrasting epidemiologic associations reported for infection exposure and B-ALL.