Hemopoietic stem cells (HSCs) are maintained at relative quiescence by the balance between the positive and negative regulatory factors that stimulate or inhibit their proliferation. Blocking the action of negative regulatory factors may provide a new approach for inducing HSCs into proliferation. A variety of studies have suggested that TGF-β negatively regulates cell cycle progression of HSCs. In this study, a dominant negatively acting mutant of TGF-β type II receptor (TβRIIDN) was transiently expressed in HSCs by using adenoviral vector-mediated gene delivery, such that the effects of disrupting the autocrine TGF-β signaling in HSCs can be directly examined at a single cell level. Adenoviral vectors allowing the expression of TβRIIDN and green fluorescence protein in the same CD34+CD38-Lin- cells were constructed. Overexpression of TβRIIDN specifically disrupted TGF-β-mediated signaling. Autocrine TGF-β signaling in CD34+CD38-Lin- cells was studied in single cell assays under serum-free conditions. Transient blockage of autocrine TGF-β signaling in CD34+CD38-Lin- cells enhanced their survival. Furthermore, the overall proliferation potential and proliferation kinetics in these cells were significantly enhanced compared with the CD34+CD38-Lin- cells expressing green fluorescence protein alone. Therefore, we have successfully blocked the autocrine TGF-β-negative regulatory loop of primitive hemopoietic progenitor cells.