This thesis examines stakeholder relationships in entrepreneurial ecosystems by focusing on the importance of sustainability, education and culture in this context. A three-staged research approach on the topic is employed. First, based on a systematic literature review the relevance of stakeholder theory for the creation of sustainable entrepreneurial ecosystems that encourage sustainable entrepreneurship is examined as conceptual approach. Second, a qualitative empirical stance is used to investigate stakeholder involvement from the entrepreneurial ecosystem at 20 higher educational institutions in 19 European countries with regard to entrepreneurship education. Third, comparative, quantitative empirical research is conducted to identify cross-cultural and stakeholder-related factors that determine the perceived strength of the sustainable entrepreneurial ecosystems in Austria and Germany. The findings of this thesis indicate that stakeholder theory, sustainability, education and culture all matter for developing strong entrepreneurial ecosystems. These results can be of interest for policy makers, entrepreneurship educaters, entrepreneurs and other entrepreneurial stakeholders, who aim at strengthening regional entrepreneurial ecosystems.