Efficient manipulation of Hedgehog (HH)/GLI signaling activity is crucial to the analysis of molecular events underlying HH/GLI-regulated cell fate determination and tumor growth. In this article, we describe the use of retroviral expression systems as a valuable tool to activate or repress Hh-pathway activity in a broad spectrum of mammalian cells-including human cells-either by forced expression of the major Hedgehog-effectors GLI1 and GLI2 or by expression of the short-hairpin RNAs-targeting GLI mRNAs. We focus on two distinct retroviral systems that allow efficient and sustainable expression of GLI proteins in primary cells and cell lines of human origin: (i) a Moloney Murine Leukemia Virus-based and (ii) an HIV-derived lentivirus expression system, which allows transduction of both dividing and quiescent cells.