Notch pathway signaling is implicated in several human cancers. Aberrant activation and mutations of Notch signaling components are linked to tumor initiation, maintenance, and resistance to cancer therapy. Several strategies, such as monoclonal antibodies against Notch ligands and receptors, as well as small-molecule gamma-secretase inhibitors (GSIs), have been developed to interfere with Notch receptor activation at proximal points in the pathway. However, the use of drug-like small molecules to target the down-stream mediators of Notch signaling, the Notch transcription acti-vation complex, remains largely unexplored. Here, we report the discovery of an orally active small-molecule inhibitor (termed CB -103) of the Notch transcription activation complex. We show that CB-103 inhibits Notch signaling in primary human T cell acute lym-phoblastic leukemia and other Notch-dependent human tumor cell lines, and concomitantly induces cell cycle arrest and apoptosis, thereby impairing proliferation, including in GSI-resistant human tumor cell lines with chromosomal translocations and rearrange-ments in Notch genes. CB-103 produces Notch loss-of-function phenotypes in flies and mice and inhibits the growth of human breast cancer and leukemia xenografts, notably without causing the dose-limiting intestinal toxicity associated with other Notch inhibitors. Thus, we describe a pharmacological strategy that in-terferes with Notch signaling by disrupting the Notch transcription complex and shows therapeutic potential for treating Notch -driven cancers.