The signalling pathways downstream of the transforming growth factor beta (TGFbeta) family of cytokines play critical roles in all aspects of cellular homeostasis. The phosphorylation and activation of p38 mitogen-activated protein kinase (MAPK) has been implicated in TGFbeta-induced epithelial-to-mesenchymal transition and apoptosis. The precise molecular mechanisms by which TGFbeta cytokines induce the phosphorylation and activation of p38 MAPK are unclear. In this study, I demonstrate that TGFbeta-activated kinase 1 (TAK1/MAP3K7) does not play a role in the TGFbeta-induced phosphorylation and activation of p38 MAPK in MEFs and HaCaT keratinocytes. Instead, RNAi-mediated depletion of MAP3K4 and MAP3K10 results in the inhibition of the TGFbeta-induced p38 MAPK phosphorylation. Furthermore, the depletion of MAP3K10 from cells homozygously knocked-in with a catalytically inactive mutant of MAP3K4 completely abolishes the TGFbeta-induced phosphorylation of p38 MAPK, implying that among MAP3Ks, MAP3K4 and MAP3K10 are sufficient for mediating the TGFbeta-induced activation of p38 MAPK.