Here, we developed a clinically translatable platelet gene therapy approach for hemophilia B. Platelet-targeted FIX (2bF9) expression was introduced by transplantation of hematopoietic stem cells (HSCs) transduced with 2bF9 lentivirus (LV). Sustained therapeutic levels of platelet-FIX expression were obtained in FIX(null) mice that received 2bF9 LV-transduced HSCs. Approximately 6-39% of the platelets expressed FIX in the transduced recipients, which was sufficient to rescue the bleeding diathesis in FIX(null) mice in tail clipping models. Sequential bone marrow transplantation demonstrated that platelet-FIX expression in the secondary recipients was sustained, leading to phenotypic correction. Notably, none of the transduced recipients developed anti-FIX antibodies after platelet gene therapy. Only one of the nine recipients developed a low titer of inhibitory antibodies (1.6 BU/ml) after challenge with rhFIX. These data suggest that platelet gene therapy can not only restore hemostasis but also induce immune tolerance in hemophilia B mice, indicating that this approach may be a promising strategy for gene therapy of hemophilia B in humans.