Background: Altered regulation of many transcription factors has been shown to be important in the development of leukemia. TWIST2 modulates the activity of a number of important transcription factors and is known to be a regulator of hematopoietic differentiation. Here, we investigate the significance in acute lymphoblastic leukemia of epigenetic regulation of TWIST2 in control of cell growth and survival and in response to cytotoxic agents.Design and Methods: TWIST2 promoter methylation status was quantitatively assessed, by COBRA and pyrosequencing assays, in multiple types of leukemia and TWIST2 expression was determined by qRT-PCR. The functional role of TWIST2 in cell proliferation, survival and response to chemotherapy was assessed in transient and stable expression systems.Results: We show that TWIST2 is inactivated in more than 50% of childhood and adult acute lymphoblastic leukemia through promoter hypermethylation and this epigenetic regulation is especially prevalent in RUNX1-ETV6 driven cases. Re-expression of TWIST2 in cell lines results in a dramatic reduction in cell growth and the induction of apoptosis in the Reh cell line. Furthermore, re-expression of TWIST2 results in increased sensitivity to the chemotherapeutic agents etoposide, daunorubicin and dexamethasone and TWIST2 hypermethylation was almost invariably found in relapsed adult acute lymphoblastic leukemia (91% of samples hypermethylated).Conclusions: This study suggests a dual role for epigenetic inactivation of TWIST2 in acute lymphoblastic leukemia, initially through altering cell growth and survival properties and subsequently in increasing the resistance to chemotherapeutic treatment.