The mechanism of neural progenitor cell differentiation is not yet clearly understood. One mechanism of differentiation control is the activation of gene transcription by beta-catenin, a key player in the canonical Wnt-pathway, whereby gene activation depends on beta-catenin translocation into the nucleus. We quantified beta-catenin signals in different cellular compartments during cell differentiation by semi-automatic quantitative analysis of confocal image stacks using Imaris™. To investigate the influence of Wnt signals on beta-catenin localization, the cells were treated with either the glycogen synthase kinase-3beta inhibitor SB216763, or WIF-I (Wnt inhibitory factor), an inhibitor of free Wnt proteins. Wnt3a, a stimulator of the canonical Wnt-pathway, was used to investigate the upstream effects of this pathway. The results show that the nuclear beta-catenin concentration increases after induction of cell differentiation. Treatment with SB216763 enhanced this effect, whereas WIF-I treatment suppressed it. The development of neuronal phenotypes was quantified in parallel to support the evidence that the differentiation is controlled by the Wnt-pathway. This precise quantification of fluorescence signals in 3D volumes correlates with neuronal phenotype development and provides a means to further study cell differentiation and protein translocation processes between cellular compartments.