<p>The entrepreneurial ecosystem concept has gained significant traction in both academic and policy discussions. However, the internal mechanisms and interrelations within and between entrepreneurial ecosystems are not fully understood: entrepreneurial ecosystem black boxes need to be opened. To advance the field, this special issue identifies key research themes essential to developing a rigorous and transdisciplinary entrepreneurial ecosystem research program. The first theme explores the configurations of entrepreneurial ecosystem elements, focusing on their structure, composition, and impact on different types of entrepreneurship. The second theme examines social interactions within and across entrepreneurial ecosystems, analyzing network characteristics, relationships, and temporal dynamics. The third theme investigates ecosystem outcomes, assessing entrepreneurial outputs and their contributions to aggregate welfare and sustainable development. The fourth theme delves into entrepreneurial ecosystem evolution, including mechanisms like entrepreneurial recycling and the feedback loops that drive ecosystem transformation. The fifth theme addresses entrepreneurial ecosystem boundaries, emphasizing the need for clear delimitation while recognizing inter-ecosystem connectivity. Finally, the sixth theme highlights the measurement of entrepreneurial ecosystems, advocating for standardized metrics to ensure comparability and the accumulation of knowledge. The special issue includes six original studies that contribute to opening these entrepreneurial ecosystem black boxes, employing diverse methodologies and addressing significant gaps in entrepreneurial ecosystem research. By fostering cumulative scholarship, this collection aims to enhance understanding and inform evidence-based policy and practice in entrepreneurial ecosystem development.</p>