Genotoxicity assessment is crucial prior to the clinical application of integrating gene therapy vectors. We developed the in vitro immortalization (IVIM) assay and the surrogate assay for genotoxicity assessment (SAGA) to predict the oncogenic risk of viral vectors. The IVIM assay primarily detects insertional mutants with integrations in or near the Mecom locus with consequent transactivation of proto-oncogenes leading to immortalization of myeloid progenitors. SAGA detects vector-associated dysregulation of gene expression due to malignant insertional mutagenesis. Both assays depend on lineage-negative cells from murine BM. In accordance with the 3R principles, we worked on expansion protocols of primary cells to reduce the number of experimental animals needed. Highly purified LSK SLAM cells expanded over 20,000-fold after two-week culture in medium containing SCF, IL3, FLT3-L and IL11 (S3F11). However, the massive expanded cell population consisted mainly of myeloid progenitors. In addition, we could maintain a more stem cell-like phenotype and further increased the total cell number by supplementing S3F11 with small compounds. A83-01, pomalidomide and UM171 (APU) decelerated myeloid differentiation and mast cell development with superior expansion. S3F11- and S3F11+APU-expanded cells were used in IVIM and SAGA. After transduction with gammaretroviral vectors containing mutagenic full-length long-terminal repeats, expanded cells showed a dysregulation of the oncogenic gene expression signature in SAGA. As molecular evidence of insertional transformation, integration site analysis identified a typical Mecom insertion in an immortalized clone.