This chapter focuses on the specific role of mid-range universities in knowledge transfer and regional development. It explores knowledge flows from these mid-range universities facing to a number of additional constraints in Central Eastern European regions. It brings cases from the non-metropolitan regions of Hungary and Czechia in order to examine the specific barriers to knowledge transfer and explain the reasons behind the traditionally weaker role of mid-range universities in innovation. The chapter concludes that not only the position of universities in the collaboration with business sector but their role in the innovation system is quite different, and there is a need for much more complex policies initiating the support of the university sector and starting the development of high-tech industries, small-scale enterprises, and constructing a regional advantage. The case studies show why the Czech regions are more successful in adopting appropriate policies and creating a more developed innovation ecosystem.