PURPOSE. Aqueous tear deficiency due to lacrimal gland insufficiency is one of the major causes of dry eye disease. In severe cases, such as Sjogren’s syndrome, Stevens-Johnson syndrome, or ocular cicatricial pemphigoid, therapy with artificial tears is often insufficient to relieve severe discomfort, prevent progressive ocular surface disease, or enable visual rehabilitation by corneal transplantation. Cell or organ generation from stem cells, resulting in tear-like secretion, presents an option as a suitable alternative treatment. To obtain deeper insights into lacrimal gland stem cells we analyzed murine lacrimal glands for markers of pluripotency, self-renewal, and differentiation. METHODS. A special, patented technique with mechanical and enzymatic digestion was used to generate high numbers of cells in vitro from murine lacrimal glands. These presumptive “murine lacrimal gland stem cells” (“mLGSCs”) can be propagated as monolayer cultures over multiple passages. By means of RT-PCR, Western blot, and immunohistochemistry, markers of pluripotency and differentiation were demonstrated. Hanging drop culture was used to build organoid bodies from mLGSCs to investigate their spontaneous differentiation in three-dimensional culture with histology, immunohistochemistry, and transmission electron microscopy methods. RESULTS. Isolated mLGSCs were cultured over more than 65 passages. Murine lacrimal gland stem cells expressed markers of pluripotency such as Nanog, Sox2, Kruppel-like factor 4 (Klf4), as well as early-lineage markers of all three germ layers. Three-dimensional culture of these cells revealed their ability to differentiate into various cell types. CONCLUSIONS. Our results suggest that mLGSCs were isolated and cultured successfully. These cells have the ability to differentiate into all three germ layers. The results provide further insights into lacrimal gland stem cell physiology for engineering of a lacrimal gland construct to treat severe cases of tear deficiency in the future.