Transfusion of red blood concentrates (RCCs) is a lifesaving procedure. Nonetheless, a number of studies have revealed that transfusion of RCC products is still associated with increased risk of serious clinical outcomes. Additionally, studies demonstrating the deleterious consequences of transfusion-related immunomodulation have had conflicting results. While many previous studies have focused on accumulation of potentially harmful immunomodulatory mediators during RCC storage, recent randomized clinical trials have failed to demonstrate benefit with fresh RCC transfusion in critically ill or hospitalized patients. Noteworthy, it has been suggested that RCC manufacturing methods, which are rarely accounted for in interventional trials, may have confounded these results. In addition, the presence of extracellular vesicles (EVs) in RCC product is an important factor that has emerged as a potential mediator of the immunomodulatory activity post transfusion. Therefore, this research has focused on investigating the impact of different manufacturing methods and extracellular vesicles on immunomodulatory activity of RCC in vitro as an approach to minimize or eliminate the parameters responsible for poorer clinical outcomes. This thesis tested the hypothesis that non-RBC generated vesicles in RCC are potent mediators of RCC immunomodulatory activity in vitro, and the characteristics of these vesicles are influenced by method of blood component manufacturing and length of RCC hypothermic storage. Investigations were conducted at several levels, from detecting and characterizing extracellular vesicles in RCC products using different approaches, to assessing the immunomodulatory activity that EVs play in vitro as a function of blood manufacturing method and storage duration. Despite the progress that has been made to understand EVs, the technical limitations and lack of standardization of procedures used in EVs characterization likely contribute to the considerable variability in the reported literature. As the biological complexity of EVs creates excessive challenges and difficulties in detecting, and characterizing these EVs, most studies do not take into account the heterogeneity of EVs in the RCC products in terms of concentration, content, size, and phenotype (cell of origin). Therefore, for this research different detection methods were used, including flow cytometry, dynamic light scattering and the novel tunable resistive pulse sensing technique to characterize the diversity of EVs in the nano and sub-micron range. Results of this work have verified that RCCs contain a mixed population of EVs and not all EVs in RCC are solely from the constituent RBCs. Furthermore, assessing the impact of the blood component manufacturing methods on quality characteristics of stored RCCs showed that blood manufacturing methods significantly influence the immunomodulatory effects of RCC supernatant on monocytes in vitro and significantly affect RBC and non-RBC EV characteristics throughout storage. Collectively these differences have the potential to impact quality and safety of RBC products. The work presented here is among the first to document a functional consequence related to RCC quality measures and EV characteristics that result from different blood component manufacturing methods. In addition, this work showed that automated washing of RCCs products can reduce the immunomodulatory activity associated RCC supernatants in vitro. This work provides a better understanding of the issues that exist with current blood products, as an aim to improve the blood component manufacturing processes and the quality of the stored RCCs. This research contributes to our understanding of the complex relationship between the storage duration, blood manufacturing method, what is in the blood bags, and transfusion-related immunomodulation. Herein a novel and strong scientific foundation for the role of blood manufacturing methods and RCC EVs in immunomodulation is discussed. The tools and methods used in this work can be used in the future studies to identify the specific factors or mediators associated with transfusion-related immunomodulation.