Haploidentical hematopoietic cell transplantation (HAPLO HCT) as therapy for hematopoietic malignancy can result in long-term survival and cure for patients who require allogeneic HCT but lack an HLA-matched donor.[1] Administration of cyclophosphamide to eliminate the alloreactive T cells entering into an activated state 3–4 days post HCT following infusion of either bone marrow or mobilized peripheral blood (PBSC)[2] grafts [3] has been shown be effective GVHD prophylaxis. Although post-transplant cyclophosphamide (PTCy) has generally expanded the use of HAPLO HCT, the success of this procedure has been hindered by high risks of graft rejection, prolonged immune reconstitution with subsequent infectious complications [4], and when combined with immunosuppressive therapy may result in an increased risk of disease relapse.[5] Preclinical and clinical studies strongly suggest that posttransplant infusion of donor gamma delta (γδ) T cells during this period of slow immune recovery could constitute an effective prophylaxis against infection and relapse in HAPLO HCT. This is because of the graft-versus-leukemia (GvL) effect mediated by the donor immune cells include not only alloreactive αβ T cells but also γδ T cells and natural killer (NK) cells. In contrast to alloreactive αβ T cells that recognize mismatched minor or major HLA antigens, γδ T cells recognize their target cells in a non-HLA restricted manner and do not initiate GVHD [6].. Indeed, γδ T cells directly recognize and respond to a variety of HLA-like stress-induced self-antigens [7, 8] and are known to both facilitate engraftment [9–12] and exhibit a strong graft-versus-leukemia (GvL) effect [13]. Indeed, a significant subset of HAPLO HCT patients who received an αβ TCD (T-cell depleted) graft was shown to have favorable homeostatic reconstitution of γδ T cells compared to that observed with patients receiving Pan-CD3-depleted grafts, a finding recently confirmed by Airoldi in children receiving haplo HSCT using αβ T-cell/CD19+ B-cell-depleted grafts.[14] Decreased relapse rate and a significant improvement in relapse-free survival has also been noted among haplo HCT patients who recovered with increased peripheral blood γδ T-cell counts,[13] a finding that was found to be durable over 7 years following BMT.[15] As there is not effective in vivo techniques to increase the γδ T-cell number, we developed a graft engineering protocol to provide a post-HAPLO HCT γδ T cell “boost” during the period of early post-HCT immune recovery. Ex vivo expansion and activation of γδ T cells is required to generate an effective cell dose as they constitute only a minor circulating lymphocyte population (< 10% of T cells). Previous attempts have shown γδ T cells manufacturing procedures to be cumbersome and generally confined to the research setting. In order to simplify procedures and widen applicability for clinical therapy, we adapted the CliniMACS Prodigy (Miltenyi Biotec; Bergisch Gladbach, GERMANY) for γδ T cells manufacturing in a closed-system cGMP (current Good Manfacturing Practice) compliant process to support our pivotal Phase I clinical trial in HAPLO HCT. An overview of the general procedure is depicted in Fig. 1. A total four healthy volunteer donors were accrued. Approximately 100 mL of apheresis product was phenotyped, and loaded into the CentriCult® chamber. Automated density gradient centrifugation and subsequent washes were performed in closed system (Fig. 1) and resuspended into OpTimizer® cell culture media (Thermo Fisher, Waltham, MA) containing Zoledronate (Novartis: