Quantitative image analysis is a powerful tool to gain deeper insights into signalling cascades and networks. Spatio-temporal changes of protein distribution and the transformation of the cellular architecture are regulated by such signalling networks like the beta-catenin dependend (canonical) Wnt-pathway where, after being activated, beta-catenin accumulates and translocates into the cell nucleus, acting as transcriptional activator. We quantified nuclear fluorescence signals by semi-automatic quantitative 3D-analysis of confocal images. We measured the active and total beta-catenin signals in vitro during the differentiation of human embryonic neural progenitor cells (ReNcell VM, derived from midbrain) into neurons and glia cells. To investigate the influence of Wnt-signalling activity on the localization of beta-catenin in vitro, the cells were treated with the glycogen synthase kinase-3ß inhibitor SB216763 and the upstream effectors WIF-I, Wnt3a, and Dickkopf-1. The results show that the nuclear beta-catenin concentration increases after induction of cell differentiation. Treatment with SB216763 and Wnt-3a enhanced this effect, whereas WIF-I and Dkk1 treatment suppressed it. The neuronal cell number correlates with the altered nuclear beta-catenin concentration. To investigate whether these results are comparable with the in vivo situation, we measured beta-catenin fluorescence signals in the cell nuclei of embryonic mouse ventral midbrain cryo-sections. The results show that this image cytometry approach can also be used in cryo-sections to quantify sub-cellular protein distribution and hence allow the comparison of protein translocation processes during neuronal differentiation in vitro and in vivo.