Analysis of the mechanisms underlying cell fates requires the molecular quantification of cellular features. Classical techniques use population average readouts at single time points. However, these approaches mask cellular heterogeneity and dynamics and are limited for studying rare and heterogeneous cell populations like stem cells. Techniques for single-cell analyses, ideally allowing non-invasive quantification of molecular dynamics and cellular behaviour over time, are required for studying stem cells. Here, we review the development and application of these techniques to stem cell research.