We have developed a self-inactivating PiggyBac transposon system for tamoxifen inducible insertional mutagenesis from a stably integrated chromosomal donor. This system, which we have named 'Slingshot', utilizes a transposon carrying elements for both gain- and loss-of-function screens in vitro. We show that the Slingshot transposon can be efficiently mobilized from a range of chromosomal loci with high inducibility and low background generating insertions that are randomly dispersed throughout the genome. Furthermore, we show that once the Slingshot transposon has been mobilized it is not remobilized producing stable clonal integrants in all daughter cells. To illustrate the efficacy of Slingshot as a screening tool we set out to identify mediators of resistance to puromycin and the chemotherapeutic drug vincristine by performing genetrap screens in mouse embryonic stem cells. From these genome-wide screens we identified multiple independent insertions in the multidrug resistance transporter genes Abcb1a/b and Abcg2 conferring resistance to drug treatment. Importantly, we also show that the Slingshot transposon system is functional in other mammalian cell lines such as human HEK293, OVCAR-3 and PE01 cells suggesting that it may be used in a range of cell culture systems. Slingshot represents a flexible and potent system for genome-wide transposon-mediated mutagenesis with many potential applications.