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Objective Whole blood has shown significant promise in the military setting for resuscitation of trauma patients at risk of massive hemorrhage. Whole blood resuscitation has been increasingly applied to civilian trauma, where hemorrhagic shock is one of the most preventable causes of death. This study describes the natural experience post-implementation of a low-titer O-positive whole blood (LTOWB) protocol for adult trauma patients who presented to our academic level 1 urban trauma center. Methodology This was a single-center descriptive analysis of adult patients who received LTOWB over 12 months, from program start in December 2022 to December 2023. All patients who received LTOWB during the 12-month study period were included. Patients initially excluded from receiving LTOWB per protocol included those under the age of 16, pulseless patients, those undergoing thoracotomy or receiving cardiopulmonary resuscitation (CPR), and women 50 years of age or younger. Each case of LTOWB administration was identified and reviewed, including the number of all blood components transfused (including LTOWB). In-hospital mortality was the primary outcome. Secondary outcomes included the total amount of blood components used and markers of coagulation. Data were collected from both a trauma registry and a blood bank registry. Duplicate and missing data were assessed by two independent reviewers. Study data were stored in accordance with institutional requirements and analyzed using appropriate statistics. Results From the initiation of the LTOWB program in December 2022 through the end of December 2023, 81 adults received LTOWB in the emergency department (ED) at our center, with an overall in-hospital mortality rate of 23.5% (19). The majority of recipients were African American males, median age 33 (interquartile range (IQR) age 24-45), with penetrating trauma. The median Injury Severity Score (ISS) on presentation was 17 (IQR 10-26). These patients received a median of two units of LTOWB (IQR 1-4 units) and a median of 4 total units of blood components (inclusive of the initial LTOWB; IQR 2-12 units) in the first 24 hours. No life-threatening transfusion reactions were observed. Conclusions During the first 12 months following implementation of an LTOWB protocol for trauma patients, 81 patients received whole blood during their initial resuscitation period for traumatic hemorrhage, with no cases of significant transfusion reactions observed within 24 hours following LTOWB administration, demonstrating LTOWB administration to be safe and feasible in trauma resuscitation. While survival bias may have skewed the impact of LTOWB implementation, assessment for improved outcomes in specific sub-groups within this cohort of patients will be better elucidated in future studies through comparative analysis.