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The goal of this paper is to understand the role of the entrepreneurial ecosystem (EE) in sustaining the development of new science-based firms (NSBFs). By developing a multiple case study in the Lyon region, France, we analyzed the different roles of ecosystem actors and their importance in the different phases of company development. To do this, we developed a longitudinal study based on qualitative interviews with nineteen companies and organizations operating in the field of biotech, pharmaceutics, and medical devices, as well as on observations and secondary data. We analyzed our dataset using the critical incident technique, capturing the causal-effect relationships between firms and the other elements of the EE. Our results define the roles of its actors, as well as the stages in which they play a key role in company development. The discussion develops a theoretical approach that links the literature on EEs with the development of science-based firms. It stresses the importance of adopting a systematic approach while analyzing EEs and provides useful insights for academics, entrepreneurs, and policymakers.