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Background We sought to develop a method for the clinical large-scale depletion of αβ T lymphocytes from mobilized peripheral stem cells, which would allow the allogeneic transplantation of a graft enriched for stem cells, natural killer (NK) cells and γδ T lymphocytes. Methods Therefore, we obtained mononuclear cells from either mobilized or non-mobilized healthy adult volunteer donors and incubated the cells with a biotinylated anti-αβ T-cell Ab and subsequently with an anti-biotin Ab conjugated with magnetic microbeads. The depletion was then performed using a CliniMACS® device. Results The median T-cell depletion was 3.9 log (range 3.5-4.1 log). The recovery of the γδ and NK cells was 92% and 80%, respectively. The recovery of CD34+ stem cells from the mobilized donors was 66%. Discussion This method had no negative influence on the in vitro colony formation of stem cells, and transplantation of αβ-depleted cells into NOD-SCID IL-2 common gamma chain knockout (NOD-scid IL2r null) mice resulted in a rapid engraftment of human myeloid and lymphoid cells. This method will allow large-scale depletion of αβ T cells from mobilized peripheral blood in clinical trials. [ABSTRACT FROM AUTHOR]