Efficacy of GOS in ameliorating GVHD is dependent on the starting community composition and metabolic capacity of the gut microbiota. Principal component analysis of microbiota community composition in mice on the day prior to transplantation (A), in vitro SCFA production capacity of mouse stool prior to transplant (B-C), survival and clinical score of treatment and control BALB/c mice sourced from Jackson Laboratories (D-E), and gut microbiota among BALB/c mice sourced from Jackson Laboratories and Taconic Biosciences and their SCFA production (F-H). GOS supplementation altered community structure (n = 15 per group, P = .04, R² = 0.080; PERMANOVA using weighted UniFrac distance, A). Mice whose diets had been supplemented with GOS harbored fecal microbiota that produced more butyrate (n = 3 replicate fermentations, P = .0082, t-test, B) and more total SCFA (n = 3 replicate fermentations, P = .024, t-test, C) than un-supplemented mice. GOS supplementation likely improved survival in Jackson mice (n = 12 per group, P = .058, log-rank test; hazard ratio = .449, Cox regression, D), but to a lesser extent than in Taconic mice (see Figure 1A). GVHD clinical score, an observational indicator of GVHD severity, was assessed weekly following transplantation for each mouse (n = 12 per group, E). Community composition was different between the two groups of mice (n = 25 mice in Jackson group, 13 in Taconic group, R² = .18, P = .002, PERMANOVA using weighted UniFrac distance at the ASV level, F), and 23 of 42 genera were found to be differentially abundant between groups (q < 0.05, Benjamini–Hochberg-corrected Wilcoxon rank-sum test implemented with ALDEx2, G). All genera are plotted, with phylum designated by color and with significantly different genera designated by labels (∗Ruminococcaceae shortened to “R.” and Lachnospiraceae shortened to “L.” in subplot G). Benjamini–Hochberg-corrected q-value cutoff of 0.05 is shown as a horizontal dotted line. A positive effect size estimate indicates higher relative abundance in Taconic mice vs Jackson mice (G).The ability of gut microbiota to produce SCFA from GOS in vitro was assessed in both groups, with the experiment repeated with three different batches of mice, with two fermentations run per iteration as technical replicates. Taconic mice produced more total SCFA than did Jackson mice (n = 3 biological replicates [technical duplicates per experiment were averaged for statistical tests], P = .034, paired t-test, H). Mean and standard error are depicted by larger points and error bars (B,C,E,H).