The gene for cystathionine beta-synthase (CBS), the enzyme producing hydrogen sulfide (H2S), located on chromosome 21, is present in extra copy in Down syndrome (DS). Dysregulation of H2S signalling has been linked to DS, but the precise underlying mechanisms are yet to be fully identified. Therefore, this study aims to establish and characterize induced pluripotent stem cell (iPSC) lines reprogrammed from peripheral blood mononuclear cells (PBMC) collected from a blood sample of a paediatric DS patient (T21) and an age-matched apparently healthy donor (Eup), and also to derive iPSC into neural progenitor cells (NPC) to study the role of H2S in early neuronal differentiation. H2S levels were measured by time-lapse imaging and flow cytometry, using the H2S fluorogenic probe SF7-AM (1 μM, 30 min) and CBS protein levels were determined by immunoblot. PBMC were reprogrammed into iPSC using Sendai virus and both cell lines were successfully established and characterized. The establishment of NPC was also successful for both cell lines. T21 cells present an increase in H2S levels, which was accompanied by a 2-fold change increase in the levels of CBS protein, in comparison to the Eup lines. Overall, we found that the cellular models established by us are appropriate to study the biology of H2S in the context of DS and will allow to subsequently proceed to a model of neuronal differentiation, so the contribution of H2S to the neurodevelopment impairment in Down Syndrome patients could be unveiled.