In the presented work, a new approach for the control of aml1/eto gene expression in t(8;21)(q22;q22)-positive acute myeloid leukemia cells has been developed. The technique is based on RNA-interference and lentiviral transduction methodology. Two new lentiviral vector sets for induction of constitutive anti-aml1/eto RNA-interference in acute myeloid leukemia cells have been developed and tested. The first set was based on the use of artificial microRNAs (miRNAs), and the second one was intended for production of short hairpin RNAs (shRNAs). It was shown that Kasumi-1 and SKNO-1 leukemia cells could be efficiently transduced with each new lentiviral vector. Moreover, the percent of modified leukemia cells evaluated in a multiplicity of infection (MOI) test exceeded 90% for Kasumi-1 and SKNO-1 cells at MOI 40 and 20, respectively. Comparative study elucidated that the anti-aml1/eto shRNA-based approach induced a stronger knock-down of aml1/eto gene in Kasumi-1 and SKNO-1 cells compared to the miRNA-based method. We assume that the proposed approach will become a handy tool for regulation of aml1/eto gene expression in both in vitro and in vivo studies of the functional and biological role of the gene.