Membrane fluidity plays an important role in many cell functions such as cell adhesion, and migration. In stem cell lines membrane fluidity may play a role in differentiation. Here we report the use of viscosity-sensitive fluorophores based on a BODIPY core, termed “molecular rotors”, in combination with Fluorescence Lifetime Imaging Microscopy, for monitoring of plasma membrane viscosity changes in mesenchymal stem cells (MSCs) during osteogenic and chondrogenic differentiation. In order to correlate the viscosity values with membrane lipid composition, the detailed analysis of the corresponding membrane lipid composition of differentiated cells was performed by time-of-flight secondary ion mass spectrometry. Our results directly demonstrate for the first time that differentiation of MSCs results in distinct membrane viscosities, that reflect the change in lipidome of the cells following differentiation.