Molecular gradients play an important role in diverse physiological and pathological phenomena such as immune response, wound healing, development and cancer metastasis. In the past 10 years, engineering tools have been increasingly used to develop experimental platforms that capture important aspects of cellular microenvironments to allow quantitative and reproducible characterization of cellular response to gradients. This review discusses the emergence of microfluidics-based gradient generators and their applications in enhancing our understanding of fundamental biological processes such as chemotaxis and morphogenesis. The principles and applications of microfluidic gradient generation in both 2D and 3D cellular microenvironments are discussed with emphasis on approaches to manipulate spatial and temporal distribution of signaling molecules.