Antiretroviral drug therapy can effectively reduce the viral load, and is associated with a degree of immune reconstitution in human immunodeficiency virus (HIV)-infected patients. However, the presence of a latent viral reservoir, the development of drug resistance, drug toxicity, and compliance problems are obstacles that impede full eradication of HIV through drug therapy. The cellular introduction of genetic elements that are capable of inhibiting HIV replication is conceptually appealing as a potential new treatment paradigm for acquired immunodeficiency syndrome (AIDS). In theory, this approach can lead to the development of regenerated hematopoiesis with cells that inhibit viral replication and are protected from the pathogenic effects of HIV. Ribozymes are catalytic RNA molecules that can efficiently and selectively cleave target RNA. By ex vivo retroviral transduction, we have introduced a HIV-1 tat gene-targeted ribozyme (RRz2) and a control construct (LNL6) into granulocyte-colony-stimulating factor (G-CSF) mobilized CD34+ hematopoietic progenitor cells (HPC). Transduced autologous CD34+ cells (an approximately equal mix of RRz2 and LNL6) were infused in 10 patients in this Phase I study. After a median follow-up of 2.5 yr, gene presence and expression were detected by a sensitive polymerase chain reaction (PCR) assay in a transduced-CD34+ cell dose-dependent manner. In this chapter, we describe general considerations related to HIV hematopoietic progenitor-cell gene therapy trial design, implementation, and safety, with an emphasis on the critical steps of this process, namely vector production and characterization, target-cell selection, transduction, final product release testing, and evaluation of vector presence.