Activating mutation of FLT3 by internal tandem duplications (ITDs) in the juxtamembrane region is the most common molecular aberration found in acute myeloid leukaemia (AML). In this study, a lentiviral vector containing two promoters achieved consistent and efficient co-expression of FLT3/ITD and GFP in transduced human CD34(+) haematopoietic stem/progenitor cells (HSPCs). When cultured in medium containing stem cell factor, thrombopoietin and FLT3 ligand (FL), FLT3/ITD-transduced cells demonstrated enhanced self-renewal and survival potential, unaffected by the withdrawal of FL. These cells retained a CD34(+)CD38(-/dim) immunophenotype, typical of HSPCs. Compared to cells transduced with a vector expressing GFP alone, FLT3/ITD-transduced HSPCs had a higher fraction of cells in active cell cycle. FLT3/ITD-transduced HSPCs were more sensitive to the induction of cytotoxicity by CEP-701, a selective FLT3 inhibitor, indicating a rapid 'addiction' to signalling through this oncogenic pathway. The FLT3/ITD-transduced HSPCs showed increased expression of Pim-1, c-Myc and Cyclin D3 (CCND3), each of which may contribute to the altered genetic programme instituted by FLT3/ITD signalling. Taken together, these results indicate that FLT3/ITD mutations may contribute to leukaemic transformation of normal HSPCs by prolonging survival, promoting proliferation and partially blocking differentiation. CEP-701 may act as a potent therapeutic agent for AML stem cells harbouring FLT3/ITD mutations.