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The development of clonal imbalance after transplantation of genetically modified hematopoietic cells is a cause of concern in the long-term follow-up of patients undergoing gene therapy for the treatment of severe or acquired hematopoietic disorders. We and others have previously described how insertional proto-oncogene dysregulation by transgene integration may provoke clonal restriction and leukemia, thus becoming a dose-limiting toxicity of gene therapy. When targeting populations enriched for or depleted from hematopoietic stem cells (HSC) in the C57Bl6 CD45 chimerism model, we found that intrinsic stem cell potential is a conditio sine qua non for the establishment of expanding insertional mutants. Mice observed for 6–7 months after co-transplantation of gene-modified cells and non-transduced fresh competitor cells were monitored in regular intervals of 6 weeks and the emergence of dominant clones was assessed by flow cytometry in combination with an LM-PCR procedure validated on mixtures of polyclonal and oligoclonal DNA. Dominant clones originating after gammaretroviral insertion in the Evi1 locus reproducibly occurred with a frequency of 1:10,000 when targeting multipotent LSK cells or short-term repopulating HSC (LSK CD34+ CD135−), but no such events were detected in the progeny of >1 million Sca1- Lin- c-Kit+ (LK) cells or ~75,000 multipotent progenitor cells (MPP, LSK CD34+ CD135+). Dominant clones originating from multipotent cells and displaying insertional upregulation of Evi1 showed greatly diminished T lymphopoiesis in vivo, formally demonstrating transforming events. Residual progeny of MPP or LK cells was detected in transplanted animals with insertional events in proto-oncogenes, but these clones were unable to expand to significant levels of hematopoiesis (>1%). Targeting HSC-enriched cell populations (LSK CD34+ CD135− or LSK CD34− CD135−), a comparison of gamma-retroviral transduction conditions in a 5 days serum-free culture period and lentiviral transduction in a 20h protocol revealed that the latter conditions significantly improved chimerism with a greatly increased clonal diversity in the first 8 weeks of repopulation. However, after lentiviral transduction clonal dominance progressively developed over an observation time of 6 months, although there was no evidence for insertional proto-oncogene upregulation as the underlying cause even when using a lentiviral vector with a strong internal enhancer-promoter capable of insertional long-distance effects. Our study suggests two important conclusions: (1) Insertional mutagenesis in gene therapy is unlikely to endow differentiating progenitor cells with (leukemogenic) stem cell potential and (2) clonal restriction developing in the long-term follow-up after transplantation of gene-modified hematopoietic stem cells is not necessarily a side effect of insertional mutagenesis, but may also reflect classical “gene marking” of a stem cell clone with a strong intrinsic potential for competitive dominance.