Acute lymphoblastic leukemia (ALL) cells reside in the bone marrow microenvironment which nurtures and protects cells from chemotherapeutic drugs. The disruption of cell-cell communication within the leukemic niche may offer an important new therapeutic strategy. Tunneling nanotubes (TNTs) have been described as a novel mode of intercellular communication, but their presence and importance in the leukemic niche are currently unknown. Here, we show for the first time that primary B-cell precursor ALL (BCP-ALL) cells use TNTs to signal to primary mesenchymal stromal cells (MSCs). This signaling results in secretion of prosurvival cytokines, such as interferon-γ-inducible protein 10/CXC chemokine ligand 10, interleukin 8, and monocyte chemotactic protein-1/CC chemokine ligand 2. A combination of TNT-disrupting conditions allows us to analyze the functional importance of TNTs in an ex vivo model. Our results indicate that TNT signaling is important for the viability of patient-derived B-cell precursor ALL cells and induces stroma-mediated prednisolone resistance. Disruption of TNTs significantly inhibits these leukemogenic processes and resensitizes B-cell precursor ALL cells to prednisolone. Our findings establish TNTs as a novel communication mechanism by which ALL cells modulate their bone marrow microenvironment. The identification of TNT signaling in ALL-MSC communication gives insight into the pathobiology of ALL and opens new avenues to develop more effective therapies that interfere with the leukemic niche.