Current regulatory guidelines require RCL testing of lentiviral vector preparations used in human clinical trials. Cells transduced ex vivo and cultured four days or more must also be tested before administering to the patient. Attenuated HIV (R8.71) can be considered as a theoretical RCL that could develop from a recombination event with wild-type HIV. Importantly, R8.71 lacks accessory proteins (vip, vif, vpr and nef) that are known to be essential for viral replication in T-lymphocytes. Based on this fact, the risk of a recombinant RCL being amplified in T-lymphocytes during an ex vivo transduction and expansion protocol is questionable. To test this possibility, we transduced human T-lymphocytes with one of two RCL-negative lentiviral vector preparations (eGFP or a chimeric antigen receptor (CAR)) that were spiked or not with R8.71 or wild-type HIV. C8166 cells served as a positive control for cells permissive to R8.71. The transduction and subsequent expansion lasted 10 days total and the experiment was performed on two independent occasions. Using day 10 supernatants from all groups, a full RCL assay (with amplification and indicator phases) was performed. Product enhanced reverse transcriptase (PERT) and P24 ELISA assays served as readouts for the RCL assay. Transduction rates were determined by flow cytometry and found to be 57-60% and 10-37% for the eGFP and CAR vector, respectively. The RCL assay results showed that all T-lymphocyte groups exposed to R8.71 were negative but those exposed to wild-type HIV were positive. C8166 cells exposed to R8.71 or wild-type HIV were positive in the RCL assay. These results suggest that a recombinant RCL that possess the wild-type HIV envelope would NOT amplify in human T-lymphocytes during a model ex vivo transduction procedure. The amplification of an arguably more dangerous VSVG-enveloped recombinant RCL is still possible; however, it could be reliably detected by quicker and less expensive assays such as qPCR to VSVG sequences.