The Ewing Sarcoma (EwS) is the second most common bone tumor in children and adolescents with the highest incidence in the second decade of life. The prognosis is poor, as the 5-year survival in absence of metastases is 65% and drops to 20% when metastases appear. The emergence of EwS in a mesenchymal stromal cell of origin is triggered by a chromosomal translocation, which in 85% of cases is t(11;22) (q24; q12), giving rise to the EWS-FLI1 fusion gene. The resulting fusion protein activates a cascade of events involving chromatin remodeling and transcriptional activity leading to a profound change in the transcriptome of the cell of origin. The network of genes involved in the early steps of the transformation may provide candidate targets for drug discovery. Among the potential candidates, Lin28B is particularly interesting. It is an RNA binding protein composed of two domains, Cold Shock and Zink Knuckles Domain and it is known to drive and maintain cell stemness. As maintenance of cell plasticity may be essential to cancer progression, Lin28B may play an important role in the evolution of divergent cancer types, including EwS. Consistent with this notion, Lin28B expression correlates with poor prognosis in Ewing sarcoma. The observations suggest that Lin28B may be a potential pharmacological target, bearing in mind that EWS-FLI-1 itself cannot be targeted because, like most transcription factors, it is an unstructured protein. The interplay between Lin28B and EWS-FLI1 is still poorly understood. In the present work we built a tool set of lentiviral vectors to investigate this functional interaction. The tools have been tested for quality control and are performing as expected. We also show that depletion of Lin28B leads to the down-regulation of EWS-FLI1 its target genes