Introduction: Resuscitation of a trauma patient requires a multidisciplinary team to perform in a dynamic, high-stakes environment. Error is ubiquitous in trauma care, often related to latent safety threats (LSTs) - previously unrecognized threats that can materialize at any time. In-situ simulation (ISS) allows a team to practice in their authentic environment while providing an opportunistic milieu to explore critical events and uncover LSTs that impact patient safety. Methods: At a Canadian Level 1 trauma centre, regular, unannounced trauma ISSs were conducted and video-recorded. A retrospective chart review of adverse events or unexpected deaths informed ISS scenario design. Each session began with a trauma team activation. The on-duty trauma team arrived in the trauma bay and provided care as they would for a real patient. Semi-structured debriefing with participant-driven LST identification and ethnographic observation occurred in real time. A framework analysis using video review was conducted by human factors experts to identify and evaluate LSTs. Feasibility was measured by the impact on ED workflow, interruptions of clinical care and participant feedback. Results: Six multidisciplinary, high-fidelity, ISS sessions were conducted and 70 multidisciplinary staff and trainees participated in at least one session. Using a framework analysis, LSTs were identified and categorized into seven themes that relate to clinical tasks, equipment, team communication, and participant workflow. LSTs were quantified and prioritized using a hazard scoring matrix. ISS was effectively implemented during both low and high patient volume situations. No critical interruptions in patient care were identified during ISS sessions and overall participant feedback was positive. Conclusion: This novel, multidisciplinary ISS trauma training program integrated risk-informed simulation cases with human factors analysis to identify LSTs. ISS offers an opportunity for an iterative review process of high-risk situations beyond the traditional morbidity and mortality rounds; rather than waiting for an actual case to generate discussion and review, we prophylactically examined critical situations and processes. Findings form a framework for recommendations about improvements in equipment, environment layout, workflow, system processes, effective team training, and ultimately patient safety.