Immunosuppressive strategies currently used in hematopoietic stem cell transplantation reliably decrease graft-versus-host disease (GVHD) rates, but also impair pathogen-specific immunity. Experimental transplant studies indicate that GVHD-initiating alloreactive T cells reside primarily in naive and central memory T-cell compartments. In contrast, virus-specific T cells comprise a more differentiated memory population. After finding that the rat sarcoma/mitogen-activated protein kinase kinase/extracellular receptor kinase (RAS/MEK/ERK) pathway is preferentially activated in naive and central memory human T cells, we hypothesized that MEK inhibitors would preferentially inhibit alloreactive T cells, while sparing more differentiated virus-specific T cells. Confirming our hypothesis, we found that MEK inhibitors including selumetinib preferentially inhibited cytokine production and alloreactivity mediated by naive and central memory human CD4⁺ and CD8⁺ T cells while sparing more differentiated T cells specific for the human herpesviruses cytomegalovirus and Epstein-Barr virus. We then demonstrated that short-term posttransplant administration of selumetinib in a major histocompatibility complex major- and minor-mismatched murine model significantly delayed the onset of GVHD-associated mortality without compromising myeloid engraftment, demonstrating the in vivo potential of MEK inhibitors in the setting of hematopoietic stem cell transplantation. These findings demonstrate that targeting memory-dependent differences in T-cell signaling is a potent and selective approach to inhibition of alloreactivity.