The metabolism of a given drug depends, not solely on a particular enzyme, but rather on a complex metabolic network. Thiopurine S-methyltransferase (TPMT) catalyzes the methylation, and thus deactivation, of 6-mercaptopurine, a thiopurine used in the treatment of acute lymphoblastic leukemia. Low TPMT activity has been associated with severe toxicity of 6-mercaptopurine. Determination of mutations in the TPMT gene before starting 6-mercaptopurine therapy constitutes a quick, simple and cost-effective strategy to individualize thiopurine dosing. However, TPMT phenotype-to-genotype correlation is not complete, indicating a need for identification of novel biomarkers. Based on our recent findings and reviewing seemingly unrelated literature reports we present a synthesis of the current understanding of factors that influence TPMT activity and consequently modulate responsiveness to thiopurine treatment. Identification and understanding of these factors is crucial for improving the efficacy and safety of acute lymphoblastic leukemia treatment.