Objective: A natural evolution to an IVF program that cultures embryos to the blastocyst stage is a desire to cryopreserve spare blastocysts effectively for future use. The aim of this study was to evaluate the outcome of thaw cycles after critical modifications were initiated in the blastocyst cryopreservation protocol. Design: A retrospective analysis of blastocyst cryopreservation and thaw cycles conducted from January 2000 to March 2003. Materials and Methods: All human preembryos were cultured under the same conditions prior to selection for freezing. A sequential media (P1/Blast, Irvine Scientific) was used for culture under standard laboratory procedures. Remaining blastocysts were cryopreserved on Day 5 or 6 after a fresh transfer on Day 3 or 5. The programmed replacement regimen remained consistent throughout the time period evaluated. The cryopreservation media was made internal and the protocol remained consistent throughout the study period (Freeze: 5% Glycerol, 10% Glycerol/.2M Sucrose. Thaw: 10% Glycerol/.2M Sucrose, 5% Glycerol/.2M Sucrose, .2M Sucrose). A comparison of blastocyst survival and pregnancy outcome was made between the patients thawed prior to the protocol modifications and those occurring after the modifications were incorporated. Two changes were made to the cryopreservation protocol in the year 2001. The first was an adjustment in selection criteria for blastocysts qualified to freeze and the second was an addition of assisted hatching after thaw. The selection criteria in 2000 required 40% blastocoel expansion or greater and healthy trophectoderm development evenly distributed across the blastocoel cavity. In 2001 the selection criteria adjusted the cavity expansion requirement to greater than 75%. In addition to the change in expansion criteria, a well defined inner cell mass was necessary to qualify for freezing. A final adjustment in the protocol in 2001 was to assisted hatch all thawed blastocysts prior to transfer. Results: In the two groups evaluated (36 cycles total), 13 cycles were treated according to the original protocol and 23 cycles were conducted with the modifications. A small group of cycles, 13%, overlapped in selection criteria between the two groups. In the original group, 34 blastocysts were thawed, 29 survived (86%), and 26 were transferred. In the modified protocol group, 60 blastocysts were thawed, 55 survived (91%) and 54 were hatched and transferred. No significant differences were noted between groups for female age, survival rate, or number of embryos transferred. However, there was a statistical difference (p = .001) related to pregnancy outcomes. The modified protocol resulted in a 60% clinical pregnancy rate and 47% clinical with fetal heart beat as compared to 0% clinical pregnancies with the original group. The implantation rate was also found to be higher in the modified group as compared to the original cycles, 42% vs 0%. Conclusion: A higher pregnancy rate was established in our blastocyst cryopreservation program when selection criteria was adjusted and all thawed blastocysts were treated to assisted hatching prior to transfer.