The influences of temperature on the fruit body induction of Flammulina velutipes were investigated. The fruit body could be induced after reducing of the ambient temperature under complete darkness, but could not be induced after light irradiation without temperature reduction. Fruit bodies formed under complete darkness elongated without pileus formation (pinhead fruit body). Protein expression patterns were investigated by two-dimensional electrophoresis, and 22 protein spots were found to be newly expressed in the mycelia and fruit bodies after reducing of the ambient temperature, but few protein spots were newly expressed in the mycelia after light irradiation without temperature reduction. Four of the spots had identical N-terminal sequences to polypeptides, Pf1, Pf3, Pfd3, and Pf6 which are previously sequenced as polypeptides expressed specifically during fruit body formation of F. velutipes.