T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk type of blood-cell cancer. We describe the improvement of a candidate therapeutic virus for virotherapy of leukemic cells. Virotherapy is based on the exclusive replication of a virus in leukemic cells, leading to the selective removal of these malignant cells. To improve the safety of such a virus, we constructed an HIV-1 variant that replicates exclusively in the presence of the nontoxic effector doxycycline (dox). This was achieved by replacement of the viral TAR-Tat system for transcriptional activation by the Escherichia coli-derived Tet system for inducible gene expression. This HIV-rtTA virus replicates in a strictly dox-dependent manner. In this virus, additional deletions and/or inactivating mutations were introduced in the genes for accessory proteins. These proteins are essential for virus replication in untransformed cells, but dispensable in leukemic T cells. These minimized HIV-rtTA variants contain up to 7 deletions/inactivating mutations (TAR, Tat, vif, vpR, vpU, nef and U3) and replicate efficiently in the leukemic SupT1 T cell line, but do not replicate in normal peripheral blood mononuclear cells. These virus variants are also able to efficiently remove leukemic cells from a mixed culture with untransformed cells. The therapeutic viruses use CD4 and CXCR4 for cell entry and could potentially be used against CXCR4 expressing malignancies such as T-lymphoblastic leukemia/lymphoma, NK leukemia and some myeloid leukemias.