There are strong indications that weak entrepreneurship also has a cultural dimension. Differences in entrepreneurial activity are therefore discussed in the light of cultural, personal and institutional factors, which interact on a regional level to generate a multifaceted entrepreneurial environment. The goal of this research is to shed more light on possible reasons for the persisting regional differences in entrepreneurial activity, especially for innovative start-ups, through the lens of cultural factors and personality traits. Regions strong in start-ups appear to have a specific entrepreneurial spirit supported by entrepreneur-friendly institutions and networking between regional actors. Therefore, this paper examines what defines the specific culture of successful start-up regions based on several branches in literature in entrepreneurship and regional research. The different start-up environments in Europe and the United States are summarily reviewed in light of these findings, showing fundamental differences. The discussion of findings focuses on the interplay of culture and personality, resulting in recommendations on ways to strengthen entrepreneurial culture.