Protocols for human pluripotent stem cell differentiation commonly yield a heterogeneous mix of cell types. To understand the source of heterogeneity at the single-cell level, it may be necessary to link final cell state to the cell’s history and initial state, for example to determine gene expression or morphogen signaling over the course of differentiation. Here we present methods to quantify and track single cells in time-lapse fluorescence microscopy during stem cell differentiation and link single-cell dynamics to the final resulting state in the same cells.