Recent genomic studies of pediatric acute lymphoblastic leukaemia (ALL) have identified recurrent mutations or deletions in the gene encoding the haematopoietic transcription factor IKAROS. In mice, Ikaros is essential for normal lymphocyte development, but how it promotes differentiation and inhibits leukemogenesis is unclear. Here we describe a novel approach combining inducible RNAi with ChIP-Seq to identify genes directly regulated by Ikaros in thymocytes and T cell leukemias in vivo. We generated transgenic mice allowing inducible inhibition of endogenous Ikaros expression in vivo using tet-regulated RNAi. This enabled Ikaros knockdown in distinct stages of thymocyte development and Ikaros restoration in leukaemias generated upon Ikaros knockdown. Using RNA-Seq, we investigated genes differentially-expressed upon Ikaros knockdown and restoration and overlayed ChIP-Seq data of genes bound by Ikaros in DP thymocytes in a novel approach to discovering direct transcription factor target genes in vivo. Direct target genes showed expression that significantly decreased upon Ikaros knockdown and increased upon Ikaros restoration (Ikaros-induced), or with reciprocal behaviour (Ikaros-repressed). Ikaros directly induces T cell receptor signalosome components, T cell integrins and regulates essential T lineage factor gene Notch1 in proliferating DN3 stage thymocytes, contrasting with direct Notch1 repression at the subsequent DP stage. Interestingly, activating Notch1 mutations are the most frequent lesions in human T-ALL, however the mechanisms behind Notch1 regulation remain unclear. Furthermore, Ikaros represses the protooncogene Myc across thymocyte subsets. Restoring Ikaros expression in T cell leukemias potently suppresses Notch1 and Myc and reverses their oncogenic expression profiles, provoking apoptosis and regression. These unique RNAi-based strategies reveal several novel mechanisms for Ikaros in controlling T lineage differentiation and oncogenesis.