Despite prophylactic immune suppression, clinically significant (Grade II–IV) acute graft-versus-host disease (aGvHD) afflicts up to 45% of pediatric patients receiving allogeneic hematopoietic cell transplantation (alloHCT). As aGvHD is responsible for nearly 20% of deaths following alloHCT, the need for better prevention and therapy for aGvHD is readily apparent. Involvement of the gastrointestinal (GI) tract in the pathogenesis of aGvHD has been substantiated by the translation of pre-clinical and clinical studies. Emerging evidence suggests that perturbations in the microbiota diversity result in aberrant systemic immune response as well as pathogen colonization and mucosal invasion, fostering the development of GvHD. Pre-clinical studies also suggest that replenishing commensals like Lactobacillus prior to HCT substantially decrease GvHD severity and intestinal insult. Our pilot data suggest that probiotics are safe to administer prior to and during children and adolescents undergoing HCT (IND#108,977). This proposal is a double-blind, randomized, multi-center intervention trial to evaluate the specific effect of probiotics in preventing GI aGvHD and more generally the effects on overall GvHD severity. The proposal is an approved concept of Children’s Oncology Group’s (COG), a NCI National Clinical Trial Network group. The study will be conducted through COG using its clinical research infrastructure. Correlative laboratory studies (plasma and stool analysis) will be performed in order to elucidate the mechanisms of action of probiotic therapy. The probiotic, Lactobacillus plantarum (LBP), or placebo will be administered to 384 evaluable children and adolescents undergoing alloHCT for hematologic malignancy beginning with the initiation of conditioning through Day 56. We hypothesize that maintaining epithelial cell integrity through the administration of probiotic therapy will lead to restoring microbial diversity, which will preserve immune tolerance and prevent aGvHD. The primary study aim is to determine efficacy of orally-administered LBP in preventing the development of GI aGvHD in children and adolescents undergoing alloHCT for the treatment of cancer. Secondary study aims are: (1) To determine whether orally-administered LBP decreases the incidence of Grade II–IV aGvHD following alloHCT; (2) To determine whether LBP administration maintains intestinal integrity as measured by mean plasma citrulline levels and reduction in mucosal barrier injury (MBI) bacteremia; (3a) To measure the effects of LBP on the intestinal flora phylogenetic composition during and after alloHCT using 16S rRNA gene deep sequencing; (3b) To measure effects of LBP on intestinal flora function during and after alloHCT using metagenomic and metabolite profiling; and (4) To measure proposed immunomodulatory effects of LBP in mean plasma levels of alloreactive-induced inflammatory cytokines (IL-2, IL-6, IL-12p70, IFNγ, and TNFα) in patients receiving LBP compared to placebo. The proposed study will be the first double-blind randomized controlled trial to evaluate the effect of probiotics in preventing GI aGvHD in pediatric HCT. If a beneficial effect is observed, expansion of the proposed intervention to other pediatric and adult malignant conditions requiring alloHCT may improve overall quality of life and prevent transplant-related complications like aGvHD.