Gene therapy (GT) approaches based on autologous hematopoietic stem cells (HSC) corrected ex vivo have shown therapeutic benefit in a number of inherited disorders. GT bares the advantage of allowing each patient to be her/his own donor while reducing the risks of immune-mediated complications as compared with allogeneic hematopoietic stem-cell transplantation (HSCT). In order to achieve stable engraftment of HSC, patients undergoing transplantation of allogeneic or autologous HSC receive a chemotherapy- and/or radiotherapy-based preparation. With regard to HSC-GT for inherited genetic disorders, the ideal conditioning regimen should aim to contain toxicity by reducing the dosage and/or the number of chemotherapeutic agents administered, in comparison to fully myeloablative preparations employed in conventional allogeneic HSCT. To meet this aim, a profound knowledge of the disease-specific biological background and of the therapeutic transgene levels, as well as of the key principles of transplantation, are required. While low-dose conditioning is sufficient to create a mixed chimerism when gene-corrected cells are endowed with a natural selective advantage, such as in the case of immune deficiencies, myeloablative doses are necessary when high levels of engraftment are required in disease such as lysosomal storage disorders and beta thalassemia. Therefore, the intensity and type of conditioning regimen administered to patients undergoing HSC-GT should be tailored to reach a minimal efficacious therapeutic target level while sparing toxicity. Novel strategies based on monoclonal antibodies selectively depleting blood cells and associated with limited extramedullary toxicity might be successfully employed in the context of HSC-GT in the near future. This review focuses on the role of the conditioning regimen in HSC-GT, and in particular, it highlights the importance of modulating the preparative chemotherapy based on disease biology and transgene expression in order to optimize outcome.