Around 20% of patients with acute lymphoblastic leukemia are Philadelphia chromosome positive (Ph-positive acute lymphoblastic leukemia) and express the Bcr/Abl tyrosine kinase. Treatment with the tyrosine kinase inhibitor Imatinib is currently standard for chronic myelogenous leukemia, which is also caused by Bcr/Abl. However, Imatinib has shown limited efficacy for treating Ph-positive acute lymphoblastic leukemia. In our study, we have investigated the effect of Imatinib therapy on murine P190 Bcr/Abl lymphoblastic leukemia cells. Three of four cultures were very sensitive to treatment with 5 mumol/L Imatinib. Significant cell death also initially occurred when the same cultures were treated in the presence of stromal support. However, after 6 days, remaining cells started to proliferate vigorously. The Bcr/Abl tyrosine kinase present in the cells that were now able to multiply in the presence of 5 mumol/L Imatinib was still inhibited by the drug. In concordance with this, the Abl ATP-binding pocket domain of Bcr/Abl in the resistant cells did not contain point mutations which would make the protein Imatinib resistant. The effect of stroma in selecting Imatinib-resistant lymphoblasts did not require direct cell-cell contact. SDF-1alpha could substitute for the presence of stromal cells. Our results show that stroma selects Imatinib-resistant Bcr/Abl P190 lymphoblasts that are less dependent on Bcr/Abl tyrosine kinase activity. Therefore, therapy for Ph-positive acute lymphoblastic leukemia, aimed at interfering with the protective effect of stroma in combination with Imatinib, could be of benefit for the eradication of the leukemic cells.