Pharmacological targeting of Janus kinase 3 (JAK3) has been employed successfully to control allograft rejection and graft-vs.-host disease (GVHD). Recent evidence suggests that in addition to its involvement in common-gamma chain (cgamma) signaling of cytokine receptors, JAK3 is also engaged in the CD40 signaling pathway of peripheral blood monocytes. In this study, we assessed the consequences of JAK3 inhibition during CD40-induced maturation of myeloid dendritic cells (DCs), and tested the impact thereof on the induction of T-cell alloreactivity. Dendritic cells triggering through CD40 induced JAK3 activity, the expression of costimulatory molecules, production of IL-12, and potent allogeneic stimulatory capacity. In contrast, JAK3 inhibition with the rationally designed JAK3 inhibitor WHI-P-154 prevented these effects arresting the DCs at an immature level. Interestingly, DCs exposed to the JAK3-inhibitor during CD40-ligation induced a state of hyporeactivity in alloreactive T cells that was reversible upon exogenous IL-2 supplementation to secondary cultures. These results suggest that immunosuppressive therapies targeting the tyrosine kinase JAK3 may also affect the function of myeloid cells. This property of JAK3 inhibitors therefore represents a further level of interference, which together with the well-established suppression of cgamma signaling could be responsible for their clinical efficacy.