The adoptive transfer of donor CD4+CD25+ regulatory T cells has been shown to protect from lethal graft-versus-host disease after allogeneic bone marrow transplantation in murine disease models. Efficient isolation strategies that comply with good manufacturing practice (GMP) guidelines are prerequisites for the clinical application of human CD4+CD25+ regulatory T cells. Here we describe the isolation of CD4+CD25+ T cells with regulatory function from standard leukapheresis products by using a 2-step magnetic cell-separation protocol performed under GMP conditions. The generated cell products contained on average 49.5% CD4+CD25high T cells that phenotypically and functionally represented natural CD4+CD25+ regulatory T cells and showed a suppressive activity comparable to that of CD4+CD25+ regulatory T-cell preparations purified by non-GMP-approved fluorescence-activated cell sorting.