RasGAP-SH3 binding protein (G3BP) is a player in the ras signal transduction pathway and is involved in mRNA turnover. G3BP is a multifunctional protein affecting cell growth, cell stress responses, and cell transformation. The goal of this study was to define ex vivo function(s) of G3BP in several systems. We developed two RNA interference methods to knockdown G3BP in murine cell lines. We observed that the partial plasmid-mediated RNAi knockdown of MmuG3BP1 does not affect muscle cell differentiation. To improve efficiency, we successfully developed lentiviral-mediated RNAi against MmuG3BP1 resulting in significant knockdown in NIH-3T3 cells. We also developed vectors for pull-down experiments designed to identify G3BP1's binding proteins. Furthermore, we generated an efficient polyclonal anti-GST-G3BP1 antibody. Thus, we developed and tested tools to define the ex vivo function(s) of G3BP1.