In health care, teamwork skills are critical for patient safety; therefore, great emphasis is placed on training these skills. Given that training is increasingly designed in a blended way, serious games may offer an efficient method of preparing face-to-face simulation training of these procedural skills. This study aimed to investigate the teamwork principles that were used during gameplay by medical students and teamwork experts. Findings can improve our understanding of the potential of serious games for training these complex skills. We investigated a web-based multiplayer game designed for training students' interprofessional teamwork skills. During gameplay, 4 players in different roles (physician, nurse, medical student, and student nurse) had to share information, prioritize tasks, and decide on next steps to take in web-based patient scenarios, using one-to-one and team chats. We performed a qualitative study (content analysis) on these chats with 144 fifth-year medical students and 24 health care teamwork experts (as a benchmark study) playing the game in groups of 4. Game chat data from 2 scenarios were analyzed. For the analysis, a deductive approach was used, starting with a conceptual framework based on Crew Resource Management principles, including shared situational awareness, decision-making, communication, team management, and debriefing. Results showed that most teamwork principles were used during gameplay: shared situational awareness, decision-making (eg, re-evaluation), communication (eg, closed loop), and team management (eg, distributing the workload). Among students, these principles were often used on a basic level. Among experts, teamwork principles were used with more open forms of speak up and more justification of decisions. Some specific Crew Resource Management principles were less observed among both groups, for example, prevention of fixation errors and use of cognitive aids. Both groups showed relatively superficial debriefing reflections. Playing a multiplayer game for interprofessional teamwork appears to facilitate the application of teamwork principles by students in all important teamwork domains on a basic level. Expert players applied similar teamwork principles on a moderately high complexity level. Some teamwork principles were less observed among both students and expert groups, probably owing to the artifacts of the game environment (eg, chatting instead of talking). A multiplayer game for teamwork training can elicit the application of important, basic teamwork principles, both among novices and experts, and provides them with a flexible, accessible, and engaging learning environment. This may create time for exercising more complex skills during face-to-face training.