Cancer-associated fibroblasts (CAFs) arising from bone marrow–derived mesenchymal stromal cells (MSCs) are prominent in B-cell precursor acute lymphoblastic leukemia (B-ALL). We have previously shown that CAF formation is triggered by exposure to reactive oxygen species–inducing chemotherapy and that CAFs support chemoresistance by donating mitochondria to the cancer cells through tunneling nanotubes. In the present study, we show that exposure of MSCs to ALL cell lines, patient-derived xenografts, and primary cells or their conditioned media can also trigger CAF formation. Using bulk RNA sequencing in cell lines, we show that the MSC to CAF transition is accompanied by a robust interferon pathway response, and we have validated this finding in primary cells. Using confocal microscopy and flow cytometry, we identify the uptake of leukemia cell–derived mitochondrial double-stranded RNA (dsRNA) by MSCs as a proximate trigger for the MSC to CAF transition. We demonstrate that inhibiting dsRNA formation in ALL cells by treatment with low-dose ethidium bromide or the mitochondrial transcription inhibitor IMT1, or degrading dsRNA in conditioned media by 100°C exposure eliminates the ability of the ALL conditioned media to stimulate MSC to CAF transition. Our data reveal, to our knowledge, a novel and previously undescribed mechanism by which cancer cells induce a CAF phenotype in stromal cells, showing how B-ALL cells can directly induce the previously described niche-mediated protection within the bone marrow.