Lentiviral vectors (LVs) have provided an efficient way to integrate our gene of interest into eukaryote cells. Human immunodeficiency virus (HIV)-derived LVs have been vastly studied to become an invaluable asset in gene delivery. This abled LVs to be used in both research laboratories and gene therapy. Pseudotyping HIV-1 based LVs, abled it to transduce different types of cells, especially hematopoietic stem cells. A wide range of tropism, plus to the ability to integrate genes into target cells, made LVs an armamentarium in gene therapy. The third and fourth generations of self-inactivating LVs are being used to achieve safe gene therapy. Not only advanced methods enabled the clinical-grade LV production on a large scale, but also considerably heightened transduction efficiency. One of which is microfluidic systems that revolutionized gene delivery approaches. Since gene therapy using LVs attracted lots of attention to itself, we provided a brief review of LV structure and life-cycle along with methods for improving both LV production and transduction. Also, we mentioned some of their utilization in immunotherapy and gene therapy.