The aim of this study was to investigate the expression of chemokine receptors on T cells and functional changes of T helper (Th) cells in peripheral blood stem cell (PBSC) harvests after treating healthy donors with recombinant human granulocyte colony-stimulating factor (rhG-CSF). Using multiparameter flow cytometry, we analyzed the expression of CXCR3 and CCR6 on T cells and the production of interferon-gamma (IFN-gamma), interleukin-4 (IL-4), and IL-17 by CD4(+) Th cells in PBSC grafts of healthy donors after in vivo rhG-CSF application. Alterations in the relative expression levels of T cell receptor beta variable (TCRBV) family members were determined using real-time polymerase chain reaction (PCR). rhG-CSF mobilization significantly decreased the expression of CXCR3 and CCR6 on T cells. Treating donors with rhG-CSF resulted in decreased IFN-gamma production and dramatically increased IL-4 and IL-17 secretion by CD4(+) Th cells, leading to T cell polarization from the Th1 to the Th2 phenotype and a preferential increase in IL-17-producing CD4(+) Th cells. We did not observe any differences in the relative expression levels of TCRBV family members before and after in vivo rhG-CSF application. Our results suggest that the expression of CXCR3 and CCR6 on donor T cells was dramatically downregulated and an IL-17 phenotype of CD4(+) Th cells was preferentially induced in PBSC grafts after treating healthy donors with rhG-CSF. The observed effects of rhG-CSF on T cells may be independent of the relative expression levels of TCRBV family members.