Externally controlled systems using an inducer are a promising strategy to improve the safety and efficacy of gene therapy and immunotherapeutic approaches. However, most inducible ON-systems that become activated by a drug require the presence of a chimeric transcription factor (transactivator), which can be toxic and impede optimal clinical translation. To address this issue, we previously developed the first all-in-one, transactivator-free lentiviral vectors (LVs) named Lent-On-Plus (LOP) that can control transgene expression in human stem cells using doxycycline (Dox). In this study, we expanded on this work by generating new versions of the LOP LVs, including different insulators, and evaluated their potential for generating inducible advanced therapy medicinal products (ATMPs), with a special focus on primary human T cells. Our results revealed that insertion of the Is2 insulator into the 3′ long terminal repeat of the LOP LVs was essential to control transgene expression in human primary T cells, which was distinct from all other cell types analyzed. Notably, the inducible primary T cells generated by the LOPIs2 LVs were responsive to ultralow doses of Dox (<100 pg/ml) and showed no changes in phenotype or function compared to untransduced T cells. Furthermore, the LOPIs2 system was effective in regulating transgene expression in vivo upon orally administration of Dox. As proof-of-concept, we validated the LOPIs2 system by generating inducible CAR-T cells that selectively kill CD19+ cells in the presence of Dox. In summary, we describe here the first transactivator-free, all-in-one system capable of generating Dox-inducible ATMPs.