Background: B cell acute lymphoblastic leukemia with overexpression of CRLF2 (CRLF2 B-ALL) is a Ph-like leukemia with poor outcome in children, as well as in adults. Activation of the CRLF2 receptor by the cytokine, TSLP, results in activation of the JAK/STAT and PI3K/AKT/MTOR pathways. Activation of these pathways has been associated with oncogenesis and chemoresistance and their downstream targets include members of the Bcl-2 family. Our objective here was to determine the effect of TSLP on expression of Bcl-2 family proteins and the efficacy of targeting Bcl-2 family pro-survival proteins in CRLF2 B-ALL. Methods: Human CRLF2 overexpressing B-ALL cell lines (MUTZ5 and CALL4) were cultured with and without TSLP and evaluated for expression of Bcl-2 family pro-survival and pro-death proteins by flow cytometry. Flow cytometry was also used to assess for Annexin V/7-AAD and Caspase 3/7 assays of apoptosis following treatment of CRLF2 B-ALL cells with the Mcl-1 inhibitor (Mim-1). Results: Our data show that TSLP induces CRLF2 B-ALL cells to increase expression of the Bcl-2 family pro-survival proteins Bcl-XL and Mcl-1, but not Bcl-2. TSLP also increased the expression of several Bcl-2 family pro-death proteins including Bim, Bak, Bax, and Bad. Treatment with Mcl-1 inhibitor (Mim-1) reduced cell counts by > 90% and this reduction was maintained in the presence of TSLP. Reductions in cell number were due to a dose-dependent increase in caspase-mediated apoptosis reaching > 95% cell death after 3 days in concentrations of Mim-1 over 15 uM, even when TSLP was present. Conclusions: These data suggest that in patients where TSLP stimulates CRLF2 B-ALL cells, leukemia cells are likely to express high levels of pro-survival molecules to sequester the high levels of pro-death molecules contributing to the survival of leukemia cells. These data also suggest that therapies which can inhibit Bcl-2 family pro-survival proteins (inhibitors of Mcl-1 and/or Bcl-XL) are likely to make CRLF2 B-ALL cells highly susceptible to apoptosis, due to the elevated levels of pro-death molecules induced by TSLP.