We simulated gene therapy using parameters derived from the analysis of autologous transplantation studies in glucose-6-phosphate dehydrogenase heterozygous cats to determine how hematopoietic stem cell (HSC) biology might influence outcomes. Simulation illustrates that a successful experiment can result by chance and may not be the repeated outcome of a specific protocol design or technical approach. As importantly, in many simulated gene therapy experiments where 1, 2, or 6 of 30 transplanted HSC were labeled, there was significant variation in the contribution from marked clones over time. Variability was minimized in simulations in which large numbers of HSC were transplanted. Strategies that may permit consistent clinically successful results are presented. Taken together, these simulation studies demonstrate that the in vivo behavior of HSC must be considered when optimizing approaches to gene therapy in large animals, and perhaps by extension, in humans.