Time-lapse video microscopy is a technique that allows one to assess cellular behavior in real-time. Two-dimensional image data is repeatedly collected at distinct time intervals, and a movie is assembled. Applications include assessment of cell migration, division, communication, growth, and death. A unique advantage to time-lapse video microscopy for assessment of these parameters is that data is collected at the single-cell level, allowing one to assess heterogeneity within a mixed population of cells. Therefore, this is particularly advantageous for cancer biology and stem cell biology. In this protocol, we describe the use of time-lapse video microscopy for (1) the real-time monitoring of cell cycle kinetics of heterogeneous populations of cancer cells, and (2) the real-time assessment of symmetric versus asymmetric stem cell division.