Background: Transfusion of cold‐stored platelet concentrates (CS‐PCs) appears effective in massively bleeding patients. However, few studies have evaluated their in vivo hemostatic function in severe thrombocytopenia. Study Design and Methods: The in vivo function of plasma‐depleted human PCs was evaluated in rabbits with a blocked reticuloendothelial system and busulfan‐induced thrombocytopenia. On day 1, a human apheresis PC was processed in a platelet additive solution (PAS‐PC) and split evenly for cold or room temperature storage (RTS). On days 3, 6, or 9, RTS‐ or CS‐PAS‐PCs were transfused (4.0 × 109 platelets/kg) after plasma depletion into two to four rabbits that developed adequate thrombocytopenia (<25 × 109/L). Ear bleeding time was measured by two incisions in small veins. The hemostatic rate was defined as the percentage of rabbits achieving bleeding cessation within 600 s at either incision. The experiment was repeated using five different PCs on each storage day. Results: The mean pre‐transfusion rabbit platelet count was 8.6 ± 5.2 × 109/L. The hemostatic rates with RTS‐ and CS‐PAS‐PCs were both 100% on day 3, 93 ± 15% and 73 ± 15% on day 6 (p =.07), and 65 ± 36% and 73 ± 37% on day 9 (p =.27), respectively, with no statistical differences. Total platelet counts were significantly lower after CS‐PAS‐PC than RTS‐PAS‐PC transfusion on all days (e.g., 58.7 ± 5.7 vs. 42.4 ± 14.7 × 109/L, p =.0007, day 9), and did not reach 50 × 109/L in several experiments. Platelet count increments correlated significantly with hemostatic efficacy for CS‐PAS‐PC transfusion only. Discussion: CS‐PAS‐PCs might achieve similar hemostasis as RTS‐PAS‐PCs in thrombocytopenic patients with mild bleeding. Hemostatic efficacy could be improved by transfusing more CS‐PAS‐PCs. [ABSTRACT FROM AUTHOR]