Granulocyte colony–stimulating factor (G-CSF) may affect T-cell homeostasis by multiple mechanisms, inducing polarization of cytokine secretion, inhibition of T-cell proliferation, and enhancement of T-cell apoptosis. We analyzed the production of interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1) by T cells from healthy volunteer donors treated with recombinant human G-CSF. Highly purified CD4⁺ T cells obtained before and after G-CSF administration (pre-G and post-G, respectively) were activated using the allogeneic mixed leukocyte reaction. Post-G CD4⁺T cells produced high levels of IL-10 but undetectable levels of IL-2 and IL-4, whereas the level of TGF-β1 release was comparable to that of pre-G CD4⁺ T cells. Notably, post-G CD4⁺ T cells proliferated poorly in response to alloantigens and to recall antigens and suppressed the proliferation of autologous CD4⁺ T cells in a cell contact–independent and an antigen-nonspecific manner. TGF-β1 and IL-10 were not dispensable for post-G CD4⁺ T cells to mediate suppression, as shown by neutralization studies. Compared with pre-G CD4⁺ T cells, alloantigen-activated post-G CD4⁺ T cells preferentially expressed markers associated with memory T cells, in conjunction with reduced levels of CD28 and CD62L. Collectively, these data demonstrate that CD4⁺ T cells exposed to G-CSF in vivo acquire the properties of T regulatory (Tr) cells once triggered in vitro through the T-cell receptor, including a peculiar cytokine production profile (IL-10⁺⁺TGF-β1⁺IL-2^low/−IL-4^low/−), an intrinsic low proliferative capacity, and a contact-independent suppression of antigen-driven proliferation. Tr cells generated ex vivo after exposure to G-CSF might be clinically relevant for transplantation medicine and for the treatment of human immune-mediated diseases.