Hematopoietic SCT (HSCT) after non-myeloablative conditioning is associated with reduced TRM, and increased risk of graft rejection. Although preclinical data have shown the importance of post transplant immunosuppression in achieving engraftment, little is known about the role of CSA in the clinical setting of non-myeloablative transplantation. In a retrospective analysis of patients treated with allogeneic HSCT after fludarabine and 2 Gy TBI, 15 of 77 evaluable patients (20%) experienced primary (n=2) or secondary graft rejection at a median of 66 days post transplant. Mean day 1-28 CSA trough levels were inversely associated with day 28 chimerism (median 99, 85 and 70% for mean CSA <300, 300-600 and >600 ng/mL, respectively; P=0.003). A similar association was observed for the cumulative incidence of graft rejection, which occurred in 8% (<300 ng/mL), 26% (300-600 ng/mL) and 50% (>600 ng/mL, P=0.005) of patients. The detrimental effect of high CSA levels on engraftment was confirmed in multivariable models and was found to operate comparably in sibling and unrelated donor transplants. Impairment of donor T-cell function by high serum levels of CSA might account for this finding, which should be verified in a larger patient group to better understand the role of CSA in non-myeloablative transplantation.