Graft-versus-host disease (GvHD) is a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation. However, the pathophysiology of GvHD remains poorly understood. In this study, we analyzed the induction of Th17 cells by monocytes of patients with GvHD in vitro, demonstrating that monocytes isolated from patients with acute skin and intestinal GvHD stage I–IV and chronic GvHD induce significantly increased levels of Th17 cells compared with patients without GvHD. S100 proteins are known to act as innate amplifier of inflammation. We therefore investigated the presence of S100 proteins in the stool, serum, and bowel tissue of patients with GvHD and the influence of S100 proteins on the induction of Th17 cells. Elevated levels of S100 proteins could be detected in patients with acute GvHD, demonstrating the release of these phagocyte-specific proteins during GvHD. Furthermore, stimulation of monocytes with S100 proteins was found to promote Th17 development, emphasizing the role of S100 proteins in Th17-triggered inflammation. Altogether, our results indicate that induction of Th17 cells by activated monocytes and the stimulatory effects of proinflammatory S100 proteins might play a relevant role in the pathogenesis of acute GvHD. Regarding our data, S100 proteins might be novel markers for the diagnosis and follow-up of GvHD.