The use of short interfering RNA (siRNA) in combination with stem cells and biocompatible scaffolds is a promising strategy in regenerative medicine. Our experimental strategy was to explore the possibility of forcing or guiding the chondrogenic differentiation of human mesenchymal stem cells (hMSCs) by knocking down a negative regulator of chondrogenesis, Slug transcription factor (TF), thus altering cell behavior. We found that TGFβ-driven chondrogenic differentiation of hMSCs cultured onto a hyaluronan-based scaffold, HYAFF(®)-11, was strengthened after cell exposure to siRNA against Slug. Slug silencing was effective in promoting the expression of chondrogenic markers, including Col2A1, aggrecan, Sox9, LEF1, and TRPS1. In addition, we confirmed that HYAFF-11 is a good scaffold candidate for hMSC use in tissue engineering applications, and showed that it is effective in sustaining TGFβ3 treatment associated with a specific gene silencing. Interestingly, preliminary results from the experimental model described here suggested that, even in the absence of differentiation supplements, Slug silencing showed a pro-chondrogenic effect, highlighting both its potential use as an alternative to TGFβ treatment, and the critical role of the Slug TF in determining the fate of hMSCs.