The chromosomal translocation t(4;11) marks infant acute lymphoblastic leukemia associated with a particularly dismal prognosis. We have recently shown that transfection of leukemic cells with MLL-AF4 siRNAs reduces leukemia-associated morbidity and mortality in xenotransplanted SCID mice suggesting that MLL-AF4 depletion negatively affects leukemia-initiating cells. In cell culture, transient suppression of MLL-AF4 expression impairs the proliferation of both t(4;11) cell lines SEM and RS4;11 more than tenfold. Clonogenicity of SEM cells was reduced fivefold and of RS4;11 cells more than twofold. One major reason for the antiproliferative consequences is the induction of apoptosis associated with caspase-3 activation and diminished BCL-XL expression. Caspase-3 was induced 3-fold and BCL-xL showed a 60% reduction after siRNA treatment compared to Mock-transfected controls in SEM cells. Analysis of intrinsic signaling pathways of apoptosis showed that MLL-AF4 depletion induces the expression of the proapoptotic genes APAF-1 and SEPT4 (ARTS). In both cell lines APAF-1 was induced more than twofold and SEPT4 more than fourfold. Our findings demonstrate that MLL-AF4 negatively interferes with an intrinsic apoptotic response and is, thus, important for the survival of these highly aggressive and chemoresistant leukemic cells. SiRNA-mediated MLL-AF4 inhibition may sensitize these cells for apoptosis-inducing agents. Therefore, complementing existing treatment strategies with a targeted inhibition of MLL-AF4 expression may lead to an improved and more specific treatment of this therapy-resistant leukemia.