Self-renewal of hematopoietic stem cells (HSCs) is key to their reconstituting ability, but the signaling pathways that regulate this process remain poorly understood. Here we show that transduction of adult mouse bone marrow cells with a constitutively activated form of Stat3 (Stat3-C) increased their regenerative activity in lethally irradiated recipients. Conversely, transduction of these cells with a dominant-negative form of Stat3 suppressed their regenerative activity. Serial transplantation and clonal tracking of the HSC progeny regenerated in vivo from STAT3-C-transduced HSCs demonstrated that the major effect of forced expression of STAT3-C was to enhance HSC self-renewal during the initial phase of hematopoietic recovery. This acquired potential for enhanced self-renewal divisions then became latent, but was reactivated when the cells were transferred to new irradiated recipients. Increased levels of activated STAT3 were also found to be associated with greater preservation of primitive hematopoietic cells in short-term cultures. These results indicate a novel biphasic regulation of HSC self-renewal in vivo in which activated STAT3 promotes HSC self-renewal under stimulated, but not homeostatic, conditions. STAT3 may thus be an important regulator of hematopoietic regeneration and a novel target for HSC engineering.