The cure rate for pediatric patients with B precursor acute lymphoblastic leukemia (pre-B ALL) is steadily improving, however relapses do occur despite initial response to therapy. To identify links between drug resistance and gene deregulation we used oligonucleotide microarray technology and determined in 184 pre-B ALL specimen genes differentially expressed compared to normal CD34+ specimens. We identified 20 signature genes including CTGF, BMP-2, CXCR4 and IL7R, documented to regulate interactions in the bone marrow. We recorded remarkably similar levels of expression in three independent patient cohorts, and found distinct patterns in cytogenetically defined subgroups of pre-B ALL. The canonical pathways that were affected are involved in inter- and intra-cellular communication, regulating signaling within the microenvironment. We tested experimentally whether interaction with stromal cells conferred protection to four drugs used in current ALL therapy, and demonstrated that bone marrow stromal cells significantly influenced resistance to vincristine and cytosine arabinoside. Compounds designed to block the identified cellular interactions within the bone marrow microenvironment are expected to mobilise the leukemic cells and make them more accessible to contemporary antileukemic agents. The data provide novel insight into the pathobiology of ALL and indicate new therapeutic targets for patients with ALL.