Early preimplantation embryos are extremely sensitive to dysregulation of cell volume, which can lead to developmental arrest. It was previously shown that mouse embryos at the 2-cell stage respond to a cell volume decrease by quickly activating Na+/H+ exchange via a signaling mechanism that involves the tyrosine kinase JAK2. However, it was not known whether this mechanism is active at the 1-cell stage, when embryos are most sensitive to perturbed cell volume. Na+/H+ exchanger activity elicited by an induced cell volume decrease was significantly lower at the mid 1-cell stage than at the late 1-cell stage or during the 2-cell stage. This activity could be completely blocked by the broad-specificity tyrosine kinase inhibitor genistein at either stage, but only at the 2-cell stage was there a substantial component of activity that was sensitive to low concentrations of the JAK2-selective inhibitors TG101348 or ruxolitinib. Western blots to detect active JAK2 that is phosphorylated on tyrosine Y1007/8 revealed that JAK2 became substantially phosphorylated in response to a cell volume decrease at the mid 2-cell, but not mid 1-cell stage. Such cell volume decrease-induced JAK2 phosphorylation appeared by the late 1-cell stage. At least in part this appears to be due to an increase in total JAK2 protein at the late 1-cell stage. Furthermore, TG101348 impaired maintenance of cell volume at the 2-cell, but not mid 1-cell, stages. Thus, cell volume homeostasis requiring Na+/H+ exchange signaled by JAK2 first becomes prominent during mouse embryonic development at the late 1-cell stage.