Phalaenopsis plantlets, acclimatized under different air temperatures (15±2, 25±2 and 35±2°C) and relative humidity (RH) (60±5, 80±5 and 95±5%RH), were transferred directly to in vivo environments for 14 days. The experiment was done at the Plant Physiology and Biochemistry Lab, National Center for Genetic Engineering and Biotechnology (BIOTEC) in year 2007. Chlorophyll a (Chla), chlorophyll b (Chlb), total chlorophyll (TC) and total carotenoid (Cx+c) contents in plantlets acclimatized under conditions of low air temperature and low RH were maintained to a higher degree than in those acclimatized under high temperature and high relative humidity by 4.45, 5.79, 4.68 and 4.95 times, respectively. Chla and TC contents in acclimatized plantlets were positively related to maximum quantum yield of PSII (Fv/Fm) (r2 = 0.61) and photon yield of PSII (ΦPSII) (r2 = 0.82), respectively. Fv/Fm, ΦPSII, photochemical quenching (qP) and transpiration rate (E) in plantlets acclimatized under low temperature and low RH were enriched and were greater than those under high temperature and high RH treatment, while stomatal conductance (gs) was lower, leading to enhanced net photosynthetic rate (Pn) and growth performances. Low temperature and low RH conditions of in vitro acclimatization should be implemented to produce healthy Phalaenopsis plantlets, defined by pigment stabilization, chlorophyll a fluorescence regulation, Pn and growth characteristics, to enable their rapid adaptation to in vivo environments.