B2-ordered Fe3Al single crystals with various orientations were deformed in tension at room temperature in vacuum. The shape of shear stress-strain curves and work hardening rates were found to be strongly dependent on the orientation. The formation of the five different work hardening stages were considered to be related to the number of operative slip systems, the effect of secondary slip systems and the dissociation of the twofold superdislocation. Stage I is an easy glide stage corresponding to single slip. Stage II, with high hardening rate, often corresponds to the existence of conjugate slip systems. Stage III, with relatively low hardening rate, corresponds to the weaker hardening of secondary slip systems. Stage IV, with the highest hardening rate, is not only related to multiple slip but also the dissociation of twofold superdislocations and the moving of superpartials with an antiphase boundary (APB) trap. Stages V, with a negative hardening rate, may be caused by the cross slip of single dissociated superdislocation. The number of stages and the work hardening rate of the same stage were also found to change significantly, when the tensile orientation lies in different orientation regions.