The deep cerebellar nuclei (DCN) are the principal output structure of the cerebellum, carrying the information sent by the Purkinje cells from the cerebellar cortex to extra-cerebellar structures. Although circuits recruiting the DCN were shown to modulate multiple motor and nonmotor functions, the mechanisms facilitating excitability and neurotransmission at their projection neurons are poorly understood. Various Transient Receptor Potential (TRP) ion channels were shown to regulate neuronal activity in the cerebellar cortex, whereas the characterization of their functional roles in the DCN is lacking. In our study, we aim to investigate the functional expression of ion channels in the mouse DCN, with a focus on the vanilloid, melastatin, and canonical TRP families. To do this, we first developed a technique of culturing postnatal mouse DCN neural cells, suitable for calcium microfluorimetry and patch clamp experiments. The excision of the cerebellar nuclei was done from parasagittal 1 mm thick slices from young C57BL/6 mice (approx. P21 - P42). After a careful dissociation of the cerebellar nuclei mass and one-week culturing, we were able to identify viable DCN neural cells, as demonstrated by evoking Ca2+ transients in response to high potassium and ionomycin solutions, respectively. Furthermore, our culturing technique proved to be suitable for screening the functional expression of TRP ion channels in DCN neurons, using a pharmacological approach. Altogether, we believe these preliminary results can further support the investigation of the basic mechanisms facilitating the cerebellar output in health and disease.