The ability of SV40 large T antigen to immortalize MEFs is largely dependent on its ability to complex with p53. Many other genes have been used to immortalize primary cells. The commonly used oncogenes may include telomerase, Kras, c-Myc, CDK4, cyclin D1, Bmi-1, and HPV 16 E6/E7, while the frequently inactivated tumor suppressor genes are p53, Rb, and p16INK, etc. Here, we demonstrate that the SV40 T antigen can effectively immortalize MEFs and the resulting piMEFs can be reversed by FLP-mediated removal of the SV40 T antigen. More importantly, our results strongly suggest that piMEFs may retain long-term proliferative activity and yet give rise to osteogenic, chondrogenic and adipogenic lineages upon BMP9 stimulation. In this study, we demonstrate that piggyBac transposon-mediated stable expression of SV40 T antigen is an efficient approach to the immortalization of primary cells. Using a retroviral vector-based reversible immortalization system expressing SV40 T antigen, we previously immortalized several types of progenitors, including MEFs, mouse hepatic progenitor cells, mouse cardiomyogenic progenitor cells, and mouse melanoblastic progenitor cells. However, the immortalization efficiency was relatively low because of the low retrovirus titers associated with the large cargo size for packaging. The piggyBac transposon system offers significant advantages over the retroviral system. First, piggyBac vector can deliver large cargo sizes, up to 100 kb of DNA fragments, into mammalian cells. Second, unlike retroviral infection, piggBac vectors can be delivered into cells with multiple copies so it is easy to achieve high levels of transgene expression. Third, liposome-based transfection is more efficient than retroviral vector-mediated infection in vitro. Fourth, piggyBac exhibits non-random integration site selectivity and has a higher preference for integrations in regions surrounding transcriptional start sites. Lastly, it is conceivable that piggyBac transposon can be removed from the host genome by its transposase and thus leaves no footprints. We are unable to remove the piggyBac vector from the piMEF genome because the wildtype transposase catalyzes the integration and excision of the transposon elements with equal efficiency. Thus, it is conceivable that the piggyBac transposon-mediated immortalization of primary cells can be reversible and footprintless. In summary, we investigate if piggyBac transposon-mediated expression of SV40 T antigen can effectively immortalize MEFs without comprising the multi-potent properties of MEFs. Using the engineered piggyBac vector pMPH86 that contains the hygromycin and SV40 T antigen expression cassettes flanked with FRT sites. Our results show that MEFs can be effectively immortalized with SV40 T antigen, and the proliferative activity of the piMEFs can be effectively reversed by FLP recombinase. The piME nonpermissive cells leading to malignant transformation.