Human and animal stem cells (rat and human adult pancreatic stem cells, salivary gland stem cells, and human dental pulp stem cells) are investigated by femtosecond laser 5-D two-photon microscopy. Autofluorescence and second-harmonic generation (SHG) are imaged with submicron spatial resolution, 270 ps temporal resolution, and 10 nm spectral resolution. In particular, the reduced coenzyme nicotinamide adenine (phosphorylated) dinucleotide [NAD(P)H] and flavoprotein fluorescence is detected in stem cell monolayers and stem cell spheroids. Major emission peaks at 460 and 530 nm with typical long fluorescence lifetimes (τ2) of 1.8 and 2.0 ns, respectively, are measured using spectral imaging and time-correlated single photon counting. Differentiated stem cells produce the extra cellular matrix (ECM) protein collagen, detected by SHG signals at 435 nm. Multiphoton microscopes may become novel noninvasive tools for marker-free optical stem cell characterization and for on-line monitoring of differentiation within a 3-D microenvironment.