In mammals, there are four Argonaute (Ago) family proteins that play crucial roles in RNA silencing, a process wherein microRNA (miRNA) mediates inhibition of target mRNA translation. Among the Ago proteins, Argonaute2 (Ago2) uniquely possesses an endoribonuclease (slicer) activity that is critical for the biogenesis of specific miRNAs and mRNA cleavage. This Ago2 slicer activity is required for postnatal development. Despite its important roles, there are still gaps in our understanding of the mechanistic basis of Ago2’s unique functions in vivo due to a limited availability of experimental tools. In order to investigate Ago2’s functions, we generated a new cellular model of Ago2-deficiency in 3T3 mouse embryonic fibroblasts (MEFs). This cell line can be used for investigating general Ago2 functions, but also for further understanding of Ago2’s unique characteristics including the slicer activity, specific amino acid residues, and domains in Ago2 by reconstitution of Ago2 mutants. Here, we describe the methods for establishing Ago2-deficient MEFs and for reconstituting the MEFs with an Ago2 mutant lacking its slicer activity by means of a retrovirus-mediated gene transfer.