The success of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is often limited by the development of acute and/or chronic graft-versus-host disease (GVHD). The lack of effective therapies to treat steroid-refractory GVHD patients has bolstered clinical evaluation of mesenchymal stromal cell (MSC) therapy for GVHD. Currently, testing of MSCs for the treatment of GVHD has exclusively used allogeneic MSCs despite emerging evidence that MSCs lose their immunoprivileged status in vivo. We hypothesized that autologous MSCs could be a viable alternative MSC source for treating active GVHD. MSCs were isolated and successfully expanded from the bone marrow of 12 volunteers (ages 2 to 55 years) who had allo-HSCT transplants and subsequently developed GVHD. MSCs from subjects with GVHD demonstrated an initial lag in growth compared with healthy control subjects; however, this lag disappeared with continued ex vivo expansion. Immunophenotype and mesodermal differentiation capacity of MSCs from GVHD patients were indistinguishable from that of healthy control MSCs. In vitro immunomodulatory functional analyses also demonstrated that GVHD MSCs were equivalent to healthy control MSCs with regards to dose dependently suppressing T cell proliferation and up-regulating indoleamine 2,3-dioxygenase expression when primed with IFN-γ. Single tandem repeat chimerism analyses further demonstrated that MSCs expanded from GVHD patients were exclusively recipient derived. Based on these data, we conclude that recipient-derived MSCs from patients with GVHD are analogous to MSCs from healthy volunteers and represent a viable option for clinical testing as an immunomodulatory option for symptomatic GVHD.