Abstract TLRs-induced maturation of dendritic cells (DC) and accumulation of inflammatory cytokines are important contributors to alloreactive T cell activation. We aimed to characterize two new MyD88-specific small molecule inhibitors, EM-163 (EM) and T923 (T9), and determine their synergism with CTLA4-Ig in a mouse skin transplant model. Both EM and T9 reduced upregulation of costimulatory molecules on DC in response to LPS. They also reduced the accumulation of IL-6 in supernatants, though the inhibition was stronger with T9 at equimolar level. Interestingly, both inhibitors promoted high MHC II expression - a favorable scenario for delivery of “Signal 1” with low “Signal 2-3” that could promote induction of T cell tolerance. In vivo analysis in a fully mismatched skin transplantation mouse model (BALB/c to B6) showed that transient administration of T9 (d0-10) combined with CTLA4-Ig doubled skin allograft survival (MST untreated: 9.5d, CTLA4-Ig only: 13d, CTLA4+T9: 19d). Overall, our results demonstrate that pharmacological inhibition of MyD88 is possible and potentiates the protective effect of costimulation blockade. Further elucidation of the mechanisms underlying this synergism and optimization of the protocol in multiple transplant models are underway. These encouraging results also suggest the use of pharmacological inhibition of MyD88 as replacement of CD154 blockade in protocols of costimualtion blockade to promote long term transplant acceptance.