The bacterial mechanosensitive channel of large conductance (MscL) directly senses and responds to membrane tension. It serves as an "emergency release valve" upon acute decreases in the osmotic environment, thus preventing cell lysis. It is one of the best studied mechanosensitive channels and serves as a paradigm of how a channel senses and responds to its membrane environment. The MscL protein is highly conserved, found throughout the bacterial kingdom, and has been shown to encode a functional mechanosensitive channel in all species where it has been studied. However, channels from different species have shown some functional variance; an extreme example is the Mycobacterium tuberculosis MscL, which when heterologously expressed in Escherichia coli requires significantly more membrane tension for gating than the endogenous E. coli MscL. We previously speculated that the membrane environment or factors not found in E. coli promoted the proper gating of the M. tuberculosis MscL channel in its native environment. Here, by reconstituting the M. tuberculosis and E. coli MscL channels in various lipids, we demonstrate that inclusion of phosphatidylinositol, a lipid found in M. tuberculosis but not E. coli, is sufficient for gating of the M. tuberculosis MscL channel within a physiological range of membrane tension.