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Introduction: Increased teammate familiarity in emergency medical services (EMS) promotes development of positive teamwork and protects against workplace injury., Methods: Measures were collected using archival shift records, workplace injury data, and cross-sectional surveys from a nationally representative sample of 14 EMS agencies employing paramedics, prehospital nurses, and other EMS clinicians. One thousand EMS clinicians were selected at random to complete a teamwork survey for each of their recent partnerships and tested the hypothesized role of teamwork as a mediator in the relationship between teammate familiarity and injury with the PROCESS macro., Results: We received 2566 completed surveys from 333 clinicians, of which 297 were retained. Mean participation was 40.5% (standard deviation [SD] = 20.5%) across EMS agencies. Survey respondents were primarily white (93.8%), male (67.3%), and ranged between 21-62 years of age (M = 37.4, SD = 9.7). Seventeen percent were prehospital nurses. Respondents worked a mean of 3 shifts with recent teammates in the 8 weeks preceding the survey (M = 3.06, SD = 4.4). We examined data at the team level, which suggest positive views of teamwork (M = 5.92, SD = 0.69). Our hypothesis that increased teammate familiarity protects against adverse safety outcomes through development of positive teamwork was not supported. Teamwork factor Partner Adaptability and Backup Behavior is a likely mediator (odds ratio = 1.03, P = .05). When dyad familiarity is high and there are high levels of backup behavior, the likelihood of injury is increased., Discussion: The relationship between teammate familiarity and outcomes is complex. Teammate adaptation and backup behavior is a likely mediator of this relationship in EMS teams with greater familiarity., (Copyright © 2017 Emergency Nurses Association. Published by Elsevier Inc. All rights reserved.)