Oxygen is a key regulator of both development and homeostasis. To study the role of oxygen, a variety of in vitro and ex vivo cell and tissue models have been used in biomedical research. However, because of ambiguity surrounding the level of oxygen that cells experience in vivo, the cellular pathway related to oxygenation state and hypoxia have been inadequately studied in many of these models. Here, we devised a method to determine the oxygen tension in bone marrow monocytes using two-photon phosphorescence lifetime imaging microscopy with the cell-penetrating phosphorescent probe, BTPDM1. Phosphorescence lifetime imaging revealed the physiological level of oxygen tension in monocytes to be 5.3% in live mice exposed to normal air. When the mice inhaled hypoxic air, the level of oxygen tension in bone marrow monocytes decreased to 2.4%. By performing in vitro cell culture experiment within the physiological range of oxygen tension, hypoxia changed the molecular phenotype of monocytes, leading to enhanced the expression of CD169 and CD206, which are markers of a unique subset of macrophages in bone marrow, osteal macrophages. This current study enables the determination of the physiological range of oxygen tension in bone marrow with spatial resolution at a cellular level and application of this information on oxygen tension in vivo to in vitro assays. Quantifying oxygen tension in tissues can provide invaluable information on metabolism under physiological and pathophyisological conditions. This method will open new avenues for research on oxygen biology.