We have engineered a lentivirus-based gene transfer system to achieve ecdysone-regulated transgene expression. The method combines the wide tropism of lentiviral vectors and the possibility of gene regulation by a small molecule with an excellent pharmacological profile. Using the hematopoietic tissue as a model, we transduced mouse progenitors with an ecdysone-regulated GFP expression cassette. The ecdysone gene switch efficiently turned GFP on and off in transplanted animals, showing low basal activity. This system allows the delivery of inducible transcriptional units in vitro and ex vivo and may be a useful tool for gene transfer purposes. Moreover, our work provides hints on the design of lentiviral vectors containing multiple expression cassettes with multiple promoters.