Virus induced gene silencing (VIGS) is a technology that has been used primarily to target the virus genome in infected plants. However, if the virus genome carries inserts derived from the host plant, the system could be employed to target the mRNAs corresponding to the host gene. Barley Stripe Mosaic Virus (BSMV), a disease of various cereals including barley and wheat, has been one of the successfully used viral RNA silencing tools in monocotyledonous plants for the last decade. In this study, we investigated several factors that play a significant role in VIGS. We tested the efficiency of silencing two genes simultaneously using the BSMV-induced gene silencing (IGS) system in detail. We found that two genes could be silenced simultaneously using BSMV-IGS. However, the silencing efficiency was found to be influenced by several factors including stability of the insert, temperature, and the accumulation of small viral RNAs from BSMV. The effect of these factors on VIGS system has been discussed.